Immunomodulating compositions from bile

ABSTRACT

The present invention relates to a composition for use as an immunomodulator comprising small molecular weight components of less than 3000 daltons, and having the following properties: a) is extractable from bile of animals; b) is capable of stimulating monocytes and macrophages in vitro; c) is capable of modulating tumor necrosis factor production; d) contains no measurable IL-1a, IL-1b, TNF, IL-6, IL-8, IL-4, GM-CSF or IFN-gamma; e) has an anti-proliferative effect in a malignant mouse hybridoma cell line; f) shows no cytotoxicity to human peripheral blood mononuclear cells; and g) is not an endotoxin. The invention also relates to a method of preparing the composition and its use an immunomodulator.

This application is a 371 of PCT/CA94/00494, filed Sep. 9, 1994, whichis a continuation of Ser. No. 08/231,726, filed Apr. 4, 1994, nowabandoned, which is a continuation of Ser. No. 08/155,303, filed Nov.22, 1993, now abandoned which is a continuation of Ser. No. 08/118,269,filed Sep. 9, 1993, now abandoned.

FIELD OF THE INVENTION

The present invention relates to immunomodulating compositions,pharmaceutical agents containing the compositions, and the use of thecompositions and agents in the treatment of animals.

BACKGROUND OF THE INVENTION

Therapies are continuously being developed for the prophylaxis andtreatment of cancer and infectious diseases, such as AcquiredImmunodeficiency Syndrome (AIDS). Some of these therapies attempt to usethe immune system therapeutically. One approach is based on the antigenspecific elements of the immune system, namely antibodies and T cells.For example, research has been aimed at developing vaccines againstforeign agents, or against certain endogenous chemical messengers, suchas interleukins, to suppress antibody reactions. A second approach isbased on the isolation, cloning, expression and production of peptidesand proteins from the non-antigen specific parts of the immune system.For example, proteins, such as cytokines, which comprise theinterleukins produced by white blood cells, and interferons whichstimulate lymphocytes and scavengers cells that digest foreign antigens,offer possibilities for therapies.

The treatment of cancer could be greatly enhanced if the early immuneresponse to a tumor could be augmented so that the tumor does not reacha critical size. Strategies which have been suggested to augment theimmune response to a tumor include vaccines specific fortumor-associated antigens; the use of monoclonal antibodies againstantigens on the surface of tumor cells such as against the interleukin-2receptor; the use of bispecific molecules containing antitumorantibodies and superantigens.

Relatively recently, the role of the physiologically active polypeptide,known as tumor necrosis factor (“TNF”) has been studied, particularlywith respect to its ability to induce necrosis of tumors, with no effectupon the normal tissues of the living body. The amino acid sequence ofTNF, as well as the base sequence of the DNA coding for TNF has beendisclosed in U.S. Pat. No. 4,879,226.

Because TNF has been shown to have a role in inducing necrosis oftumors, any agent that can stimulate the production or bioavailabilityof TNF in vivo has potential utility as a treatment for various tumorousconditions. Additionally, any agent that can stimulate human monocytesand macrophages to produce TNF in vitro, is useful as a means forproviding a source of TNF for therapeutic administration, as well as foranalytical and diagnostic purposes.

Bile, which is secreted by the liver and stored in the gall bladder, hasbeen investigated for various purposes, including the use of bileextracts to enhance bioavailability of drugs that are readilymetabolized by normal liver function (see WO 90/12583) and to inhibitleucocytosis promotion in a mammal (see Shinoda et al., Chem. Pharm.Bull., 30, 4429-4434 (1982)). However, bile has never been considered tobe a source of therapeutically useful compositions with respect toneoplastic or infectious diseases. Interestingly, in accordance withBritish Patent No. 337,797, it was suggested to use the gall bladderitself as a potential source of anti-cancer agents, but only after thebile had been removed from the gall bladder, and the gall bladderthoroughly washed.

SUMMARY OF THE INVENTION

It has now been discovered that bile is an important source of acomposition that can stimulate TNF production both in vitro and in vivoand is effective in treating various carcinomas, especially pancreaticcancer.

The bile composition of use is obtained by extraction of bile with awater soluble or miscible solvent. The extract so obtained may befurther processed to remove unnecessary or undesirable componentstherefrom.

The product obtained by the process of extracting bile disclosed infurther detail hereinbelow has been found to have TNF stimulatingactivity and is believed to have anti-cancer activity, especiallyagainst pancreatic and other cancers. Obviously, the entire compositionso obtained may not be necessary, to obtain such activity. Accordingly,it is possible to further separate, fractionate, or otherwise processthe product thus obtained, and still retain the desired TNF stimulatoryand anti-cancer activity. Moreover, it is envisioned that it is possibleto obtain synthetically a product with the same or similar TNFstimulatory and anti-cancer activity. Thus, it is envisioned that thecomponents of the product may be analyzed as to the components, orcombination thereof, that are responsible for the desired activity, anda synthetic product made, based on such analysis.

In one aspect, the present invention relates to a composition for use asan immunomodulator comprising small molecular weight components of lessthan 3000 daltons, and having one or more of the following properties:

a) is extractable from bile of animals;

b) is capable of stimulating monocytes and macrophages in vitro;

c) is capable of modulating tumor necrosis factor production;

d) contains no measurable level of IL-1α, IL-1β, TNF, IL-6, IL-8, IL-4,GN-CSF or IFN-gamma;

e) has an anti-proliferative effect in a malignant mouse hybridoma cellline;

f) shows no cytotoxicity to human peripheral blood mononuclear cells;and

g) is not an endotoxin.

In accordance with a preferred embodiment the composition is extractedfrom the bile of bovines and is capable of stimulating the release oftumor necrosis factor.

The composition of the invention may be prepared by (a) mixing bile froman animal, preferably a bovine, with a solvent that is soluble ormiscible with water, preferably an alcohol, and preferably with an equalvolume of an alcohol, to produce a bile/alcohol solution; (b) separatingthe solution which preferably is an alcohol soluble fraction, andisolating therefrom a solution substantially free of alcohol, as byremoving most of the alcohol, such as by the use of heat; (c) removingbile pigments from the solution to obtain a colorless liquid; (d)optionally treating the colorless liquid to substantially remove anyresidual alcohol; (e) removing fatty organic materials, as by extractingthe colorless liquid with ether and isolating the aqueous phase; and (f)optionally removing residual ether from the aqueous phase.

The composition may be used without further modification by simplypackaging it in vials and sterilizing. The composition may be also beused in a concentrated form. A preferred concentrated form is preparedas follows. Prior to step (e) the colorless liquid may optionally beconcentrated to about one eighth of the volume of the bile/alcoholsolution and after step (f) the aqueous phase may be concentrated sothat it is one tenth of the volume of the bile/ethanol solution.

The invention also relates to a pharmaceutical agent comprising thenovel composition of the invention.

The invention further relates to a method of treating a patientcomprising administering to said patient an effective amount of acomposition of the invention. The invention still further relates to theuse of a composition of the invention in the prophylaxis and treatmentof diseases and conditions requiring modulation of the immune response;preferably infectious diseases and neoplasias.

These and other aspects of the present invention will become evidentupon reference to the following detailed description and attacheddrawings. In addition, reference is made herein to various publications,which are hereby incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

Further details of the invention are described below with the help ofthe examples illustrated in the accompanying drawings in which:

FIG. 1 is an HPLC profile for a concentrated composition of theinvention;

FIG. 2 is an HPLC profile for a concentrated composition of theinvention;

FIG. 3 is an HPLC profile for a concentrated composition of theinvention;

FIG. 4 is an HPLC profile for a composition of the invention;

FIG. 5 is an HPLC profile for a composition of the invention;

FIG. 6 is an HPLC profile for a composition of the invention;

FIG. 7 is an HPLC profile for a composition of the invention;

FIG. 8 is a graph showing the effect of the composition on LPS-inducedrelease of TNF by PBMN;

FIG. 9 is a bar graph showing the effect of the composition onLPS-induced release of TNF by PBMN;

FIG. 10 shows the C₁₈ RP-HPLC profile of a composition of the invention;

FIG. 11 shows the RP-HPLC analysis of precipitated fractions of thecomposition of the invention;

FIG. 12 shows the RP-HPLC analysis of soluble fractions of thecomposition of the invention.

FIG. 13 is a graph showing survival taken from diagnosis of pancreaticcancer patients treated with the composition of the invention;

FIG. 14 is a graph showing survival taken from treatment of pancreaticcancer patients treated with the composition of the invention;

FIG. 15 is a graph showing survival of all melanoma patients treatedwith the composition of the invention;

FIG. 16 is a graph showing survival of melanoma patients with two ormore tumor sites treated with the composition of the invention;

FIG. 17 is a graph showing survival of melanoma patients with three ormore tumor sites treated with the composition of the invention;

FIG. 18 is a graph showing the RP-HPLC profile of whole composition ofthe invention;

FIG. 19 is a graph showing the RP-HPLC profile of a precipitate of thecomposition of the invention;

FIG. 20 is a graph showing the RP-HPLC profile of a supernatant of thecomposition of the invention;

FIGS. 21A and 21B show the conditions and times of elution of thecomposition of the invention on hydrophilic HPLC (a) and the elutionprofile for a supernatant of the composition of the invention (b);

FIG. 22 shows the elution of a precipitate of the composition of theinvention on hydrophilic HPLC;

FIG. 23 is a graph showing dose response of the composition of theinvention in stimulating peripheral blood monocyte function.

DETAILED DESCRIPTION OF THE INVENTION

As hereinbefore mentioned, the present invention relates to acomposition for use as an immunomodulator comprising small molecularweight components of less than 3000 daltons, and having the followingproperties:

a) is extractable from bile of animals;

b) is capable of stimulating monocytes and macrophages in vitro;

c) is capable of modulating tumor necrosis factor production;

d) contains no measurable level of IL-1α, IL-1β, TNF, IL-6, IL-8, IL-4,GN-CSF or IFN-gamma;

e) has an anti-proliferative effect in a malignant mouse hybridoma cellline;

f) shows no cytotoxicity to human peripheral blood mononuclear cells;and

g) is not an endotoxin.

More particularly, investigations have shown that the at least some ofthe compositions of the invention will stimulate normal monocytes toeffect cytotoxicity towards the Chang hepatoma cell line, which is usedto measure monocyte toxicity. Monocytes and macrophages from cancerpatients (cervical and ovarian cancer) also have been reported to bestimulated by the composition to attack and destroy their own particulartumor cells.

The composition of the invention can modulate tumor necrosis factor(TNF) production. A preferred composition of the invention isolated frombile from bovines, promotes the release of TNF from human peripheralblood mononuclear cells in what appears to be physiological quantities.Because TNF is known to initiate a cascade of inflammatory and antitumorcytokine effects, the preferred composition could exert itsantineoplastic effect by stimulating human leucocytes to release TNF(and possibly other cytokines). Accordingly, the present invention alsomay enhance lymphocyte and macrophage cytotoxicity towards tumor cells.

The composition of the invention has also been found to inhibit thegrowth of cells of mouse hybridoma cell line #6-1. The inhibitory effectof the composition in the mouse hybridoma cells suggestsantiproliferative activity.

The effect of the composition on the survival of human peripheral bloodmononuclear cells (PBMN) was also examined. The composition was found tobe non-cytotoxic to human PBMN.

As further exemplified below, the composition of the present inventionhas, among others, the following characteristics:

1) The component or components responsible for TNF-release from PBMNeluted early from a C₁₈ RP-HPLC column.

2) The TNF-releasing component(s) is (are) precipitated, in part, by 80%acetonitrile.

3) The material unprecipitated by 80% acetonitrile retains someTNF-releasing activity.

4) The TNF-releasing activity in both the 80% acetonitrile precipitateand supernatant fractions eluted at the same early time from RP-HPLC.The results suggest that active TNF-releasing components in thecomposition belong to the same molecular family with perhaps some subtlemolecular differences that account for solubility differences.

5) The composition causes the release of interleukin-1β (IL-1β), and thecomponent responsible for the IL-1β release elutes early from RP-HPLC,suggesting that it is likely the same substance(s) that releases TNF.

6) The composition also causes the release of low quantities ofinterleukin-2 (IL-2).

7) The composition causes the release of granulocyte macrophage colonystimulating factor (GM-CSF); the 80% acetonitrile precipitate fractionis more active than the supernatant fraction.

8) The ratio of TNF to GM-CSF release is about 2:1.

9) The 80% acetonitrile precipitate fraction contains component(s) thatrelease about 3 fold more TNF and GM-CSF than component(s) in thesupernatant fraction.

10) Analysis of the aforementioned precipitates and supernatantfractions separated by RP-HPLC shows that releasing activity for TNF,IL-1β and GM-CSF elutes early for both the precipitate and supernatant.However, in the supernatant, some IL-1β activity elutes late.

11) It is likely that the same molecule(s), i.e., component(s), in thecomposition are responsible for releasing TNF, IL-1β and GM-CSF. It ispossible that the composition acts to stimulate the release of multipledifferent cytokines, or alternatively, the composition triggers theproduction and release of one cytokine that in turn stimulatesproduction and release of other cytokines.

12) Physicochemical analysis of the composition, including theprecipitates and supernatants thereof, by SDS gel electrophoresis andmolecular sieve HPLC indicates that the principal components are lessthan 2500 daltons.

13) Further physicochemical separation by hydrophilic (polyhydroxyethyl)molecular sieve HPLC confirms the small molecular weight of thecomponents in the composition.

14) Amino acid analysis before and after acid hydrolysis suggest thepresence of peptide bonds, indicating the presence of peptides.

15) Amino acid content of the active fraction from RP-HPLC shows highlevels of glutamate/glutamine and glycine. In addition, residues ofasparagine, threonine, serine and alanine were detected.

16) There are some unidentified ninhydrin positive residues that arelikely free amino acids.

As hereinbefore mentioned, the composition of the invention may beprepared by (a) mixing bile from an animal, preferably a bovine, with anequal volume of an alcohol to produce a bile/alcohol solution; (b)separating out the alcohol soluble fraction and isolating a solutionsubstantially free of alcohol; (c) removing bile pigments from thesolution to obtain a colorless liquid; (d) treating the colorless liquidto substantially remove any residual alcohol; (e) extracting thecolorless liquid with ether and isolating the aqueous phase; and (f)removing residual ether from the aqueous phase.

The composition is obtained from the bile of any animal which producesbile, preferably non-human animals. While the composition may possess adifferent activity toward a specific disease if obtained from the bileof one species as opposed to another, a generally suitable source ofbile is that taken from bovines, ovines and swine. In most cases, it ispractical to obtain the bile of slaughtered healthy food animals, suchas bovines, ovines and pigs, for use in the preparation of thecomposition of the invention. The bile thus collected should comedirectly from the gall bladders of the slaughtered animals and should besubstantially clear, thereby indicating that the bile preparation has alow mucus content and is substantially free of pus or blood.

In a preferred embodiment of the method, bile from bovine sources isutilized. Bovine bile is plentiful, because, in part, relatively largequantities can be extracted from each animal. Moreover, bovines areroutinely slaughtered and inspected under health-related regulations,thus such animals provide a reliable source for preparing thecomposition of the invention. Furthermore, humans are less likely tohave an allergic reaction to material of bovine origin.

The bile is mixed with an equal volume of an alcohol to produce abile/alcohol solution, which is 50% alcohol. The alcohol may be analiphatic alcohol, preferably methanol, ethanol, or propanol, mostpreferably ethanol.

A solution that is substantially free of the 50% alcohol-insolublematerial may be isolated by centrifuging. Preferably, the bile/alcoholmixture is centrifuged at 3000-5000 RPM, most preferably 4200 RPM, forat least 2 hours, at about 15-25° C. The alcohol contained in thebile/alcohol-soluble fraction then may be removed by taking advantage ofthe different volatility of alcohol and water, using conventionalmethods, i.e., heating the fraction to a suitable temperature, e.g.,80-85° C., for a suitable amount of time, e.g., up to about 10 hours.

Bile pigments may be removed from the solution to obtain a colorlessliquid by using activated charcoal, polyamidic microgranules, orfiltration. Preferably, an activated charcoal treatment is utilised. Theprocedure may be repeated in order that the solution satisfies opticaldensity and conductivity standards.

The colorless liquid is treated to remove substantially any residualalcohol, using conventional methods. Preferably the colorless liquid isfiltered using a filter having about a 1.0-3.5 μm retention, mostpreferably a retention of 2.5 μm.

The colorless liquid is then extracted with ether and the aqueous phaseis isolated. The ether used in this step is preferably dimethyl ether,ethyl ether, n-propyl ether, isopropyl ether, or n-butyl ether, mostpreferably ethyl ether.

Residual ether may be removed from the aqueous phase by, for example,heating the solution up to 55° C., preferably up to about 40° C. forabout 5-15 hours, most preferably for about 10 hours.

The composition may be used without further modification simply bypackaging it in vials and sterilizing. The composition also may be usedin a concentrated form. A preferred concentrated form is prepared asfollows. Prior to step (e) described hereinabove, the colorless liquidoptionally may be concentrated to about one eighth of the volume of thebile/alcohol solution by, for example, heating to a temperature of lessthan about 85° C., preferably, to about 60°-70° C. After step (f), theaqueous phase may be concentrated so that it is one tenth of the volumeof the bile/ethanol solution by, for example, heating to about 80-85° C.

In a preferred method to prepare a composition of the invention, thecollected bile is mixed with an equal volume of ethyl alcohol. Thebile/alcohol mixture is then centrifuged at about 4200 RPM for at least2½ hours, at about 20±2° C. The supernatant liquid is decanted andchecked for pH and ethanol content. Bile pigments are then removed usingactivated charcoal. The treated bile/ethanol solution is then monitoredfor optical density (O.D.) and conductivity. O.D. levels or conductivitylevels outside acceptable specifications require that the bile/ethanolsolution be given additional treatment to remove bile pigments, forexample treatment again with activated carbon to achieve a readingwithin specification limits.

Following activated carbon treatment, the solution is filtered through afilter having a 2.5 μm retention, the alcohol is evaporated off byheating to less than 85° C. and the solution is concentrated toapproximately one eighth of the original bile/ethanol solution volume.The concentrated solution is cooled to between about 20-25° C. Thissolution is then mixed with ethyl ether and the ether phase isdiscarded. Preferably, relatively small volumes of ether and strongagitation are used, such as 0.1 to 1 volume, preferably 0.2 to 0.5volume. This step may be repeated once. The aqueous phase is heated toremove residual ether by heating up to 55° C. for about 10 hours, andfurther reduced in volume to one tenth of the original bile/ethanolvolume by heating to about 80-85° C. This solution is then tested forappearance, biological activity, and ethanol and ether content.

The pH of the composition may be adjusted to physiological pH, i.e.7.4-7.5, using hydrochloric acid (1%) solution and sodium hydroxide (1%solution), and a buffered solution may be obtained using dibasic andmonobasic sodium phosphate salts as buffers, using conventional methods.

The composition may be used without further modification by simplypackaging it in vials and sterilizing. A preferred sterilization methodis to subject the composition to three sterilization cycles byautoclaving followed by incubation.

The composition may be used in a concentrated form. The preparation ofthe concentrated form is described above. The composition may also belyophilized.

The composition and concentrated composition are clear yellowishsolutions essentially free of foreign matter, containing not more than10 ppm ethanol and not more than 5 ppm ether. In the bioassay describedin Example 4, the composition has been shown to cause non-proliferativegrowth at about 18 units per ml.

The compositions of the invention can be produced in a consistentlyreproducible form using the method as generally described above withdemonstrated identity, potency and purity from batch to batch. Identityand purity are determined using reverse-phase high pressure liquidchromatography. (See Example 1). The compositions of the invention havea consistently reproducible pattern on reverse-phase HPLC, in whichpeaks are seen early in the exclusion fraction at about 27 and 32minutes. Before, in-between and after the tall peaks, there are smallerpeaks that vary in intensity. The HPLC readings for three lots of theconcentrated composition of the invention are shown in FIGS. 1 to 3.RP-HPLC profiles for batches BO211 (FIG. 4), BO209 (FIG. 5), B29/3006(FIG. 6) and B15/1606 (FIG. 7), also show a very reproducible pattern.The compositions also display non-proliferative growth of about 18 unitsper ml in the bioassay described in Example 4. The compositions are alsocharacterized by the properties hereinbefore mentioned, for exampletheir ability to stimulate monocytes and macrophages in vitro, etc.

Compounds likely to be present in the present composition, consideringthe source, include sulfonated bile acids, oxidized bile acids, othernaturally occurring bile acids, and their amino acid (especially glycineand taurine) conjugates and sterols. Accordingly, it is believed thatthe present composition includes at least one compound having theformula

wherein the molecule may or may not be fully saturated, such that, forexample, the bond between A and B, B and C, or C and D may be single ordouble bonds, and wherein X is H, OH, ═O, or OSO₃H; and Y is

wherein R is an amino acid residue, such as, for example, glycyl,glutamyl, or tauryl, thereby forming the glycine or taurine conjugate.

In particular, the composition of the present invention has beenanalyzed as to its component compounds, including organic and inorganiccomponents. Such information was derived using standard methods ofanalytical chemistry, including mass spectroscopy (MS). The results ofsuch studies include, for example, the identification of specific bileacid compounds thought to be present, including cholic acid, glycocholicacid, deoxyglycocholic acid, ursodeoxycholic acid, cholesterol sulfate,deoxycholic acid, chenodeoxycholic acid, and taurocholic acid.

From the MS it is not distinguishable if the loss of OH and H₂ of somecompounds are occurring in the MS or if the deoxy, dideoxy andunsaturated analogs of such compounds are also present to begin with.These compounds may all be present as salts of ammonium, aklylammoniumand inorganic cations.

The MS analysis also supports the identification in the presentcomposition of phospholipids, sphingolipids and related agents capableof forming miscelles. Specific compounds thought to be present include:

stearic acid CH₃(CH₂)₁₆COOH,

palmitic acid CH₃(CH₂)₁₄COOH

oleic acid Z-9 octadecanoic acid: CH₃(CH₂)₂CH₂CH═CHCH₂(CH₂)₆COOH

oxidized or hydroxylated/unsaturated short chain fatty acids: C₆H₈O₃(e.g., CH₃CH═CHCOCH₂COOH or a C₆ acid with 2 double bonds and ahydroxide)

acetic acid

stearic acid diglyceride

palmitic acid diglyceride

stearic acid, palmitic acid diglyceride

stearic acid-monoglyceride-phosphocholine (a lysolecithin)

stearic acid monoglyceride

stearic acid triglyceride

palmitic acid monoglyceride

phosphocholine

phosphoserine

phosphosphingosine

stearic acid-sphingosine

sphingosine

stearic acid amide

stearic acid methylamide

palmitic acid amide

lecithin

sialic acid-glycerol dimer

In addition, preliminary HPLC and titration evidence has been obtainedwhich shows that shorter chain fatty acids are also present.

Phospholipid, sphingolipid, and related hydrolysis product compoundslikely to be present considering the source and the information derivedfrom the MS and HPLC analyses include at least one compound having theformula

where R′, R², R³ are different or the same and are H, COR⁴, CH═CH—R⁵, X,—P(O)(OH)O—, or —S(O)₂—; X is selected from the group consisting ofcholine, ethanol amine, N-alkylated ethanolamines, serine, inositol,sugars bearing free hydroxyls, amino-sugars, sulfonated sugars, andsialic acids; R⁴ is C₁-C₃₀ alkyl that is saturated or unsaturated,oxidized or hydroxylated; and R⁵ is an alkyl group or oxidized and/orhydroxylated analogs thereof.

The fatty acids and their conjugates may be present in theaforementioned aqueous extract as salts. The solubility of suchcompounds is also enhanced by other components of the mixture. Amides ofthe included carboxylic acids, RCONR′R², where R′ and R² are the same ordifferent and are H or alkyl, are also believed to be present.

A third class of compounds, namely, mucin and proteoglycan hydrolysisproducts, are also likely to be present, considering the source of thecomposition and the aforementioned MS analysis thereof. Such compoundsinclude hydrolysis products of mucoproteins from bile and from thegallbladder wall, such as: chondroitin 4- and 6-sulfates, dermatansulfate, heparin, heparin sulfate, hyaluronic acid and the hydrolysisproducts (monomers, dimers, oligomers and polymers) of these mucins.Chitin and other mucins may be similarly hydrolyzed, which hydrolysisproducts would include:

N-acetyl-D-glucosamine, N-acetyl-D-galactosamine-4-sulfate,galactose-6-sulfate, N-acetyl-D-glucosamine-6-sulfate,glucosamine-6-sulfate, D-glucosamine 2-sulfate, D-glucosamine2,3-disulfate, D-galactose-6-sulfate, glucuronic aid 2-sulfate,N-acetylneuraminic acid, sialic acid, N-acetyl chondrosine, chondroitin4-sulfate, chondroitin 6-sulfate, D-glucosamine, D-galactosamine,glucuronic acid, glucose, galactose, mannose, fucose, iduronic acid,hexose, hexosamine, ester sulfate, glucuronic acid, chondrosamine,2-amino-2-deoxy-D-galactose, serine, proline, threonine, alanine glycinetaurine, glutamic acid, aspartic acid, histidine, and small peptides.

Similar products would be obtained by hydrolysis of mucins such askeratin sulfates, dermatan sulfates the natural sugar-sugar linkages inthe dimers, oligomers and polymers may be replaced by —O—Si(OH)₂—O—bridges between the sugar monomers or adjacent sugar chains.

In particular, specific mucin and proteoglycan hydrolysis productcompounds thought to be present include:

sialic acids and their mono and diacetylated and glycolylated monomers;

N-acetylneuraminic acid;

hexosamines;

L-fucose;

hexosamine-hexuronic acid (dimer) disulfate;

glucuronic acid or iduronic acid disulfate, monoacetylated;

sialic acid-glycerol (dimer); and

dimers, trimers, oligomers and polymers of the above monomers inacetylated and sulfated form.

A fourth class of compounds, namely fat-soluble vitamins, likely to bepresent considering the source and the aforementioned MS analysis,include A, D, and K vitamins (e.g., D1, D3, D4, K1, K2, K5, K6, K7,K-S(II), and Vitamin E acetate, for example.

In particular, specific fat-soluble vitamin compounds thought to bepresent include at least one of the group consisting of Vitamin A2,Vitamin D1, Lumisterol (present from its vitamin D1 complex), Vitamin E,Vitamin K1 oxide, and Vitamin K5.

Various miscellaneous organic compounds are likely to be present,considering the source and the aforementioned MS analysis, suchcompounds include:

bilirubin, and its gluconuride conjugate;

biliverdin, and its gluconuride conjugate;

traces of steroids;

other plasma solutes, such as sugars, purines and pyrimidines;

miscellaneous dietary lipids; and

glutathione and its hydrolysis products.

In particular, specific miscellaneous organic compounds believed to bepresent in the composition include at least one of the group consistingof urea, methyl amine, dimethylamine, ethylamine, methylethylamine,diethylamine, dipropylamine, butylethylamine, ammonia, choline, taurine,glutamic acid, glycine, alanine, p-ser, p-eu, p-ea, asp thr ser sar,a-aba, cit, val, ile, leu, B-ala, G-aba, OH-lys, orn, lys, butylatedhydroxy toluene (BHT), and polyethylene glycol.

Amines present in the present composition, particularly the secondaryamines, may include nitrogen oxides from the air, thus forming nitrosocompounds. N-oxides and N-carbamate byproducts may also be included.This series of amines cited above should be extended to include allprimary, secondary and tertiary alkylamines.

Certain inorganic elements have been identified and quantified (mg/l) asfollows:

Tungsten 0.07 Zinc 0.666 Phosphorus 378 Cadmium 0.01 Cobalt 0.008 Nickel0.022 Barium 0.032 Iron 0.022 Manganese 0.039 Chromium 0.060 Magnesium7.46 Aluminum 0.136 Calcium 5.97 Copper 0.087 Titanium 0.01 Strontium0.060 Sodium 9600 Potassium 483 Chloride 15400 Ammonia 218 Vanadium 1ppm

The compositions of the invention have valuable pharmacologicalproperties. In particular, the compositions of the invention haveanti-proliferative effects, effect neoplastic growth, and effect releaseof tumor necrosis factor. The compositions have been shown to cause nosignificant toxicity and only transient adverse side effects (forexample, slight fever, polydipsia, pain at injection site). They havealso been found to contain no detectable components of high molecularweight matter (i.e., above about 5,000 daltons), which can cause harmfulimmunologic reactions. The compositions may be used as agents for theprophylaxis and treatment of conditions requiring modification of theimmune response, in particular infectious diseases, neoplasias, andautoimmune diseases. They may be especially useful in the treatment ofvarious forms of neoplasia, such as leukemias, lymphomas, melanomas,adenomas, sarcomas, and carcinomas. In particular, the composition maybe useful for treating malignant melanoma, pancreatic cancer,cervico-uterine cancer, cancer of the kidney, stomach, lung, rectum,breast, bowel, gastric, liver, thyroid, neck, cervix, salivary gland,leg, tongue, lip, bile duct, pelvis, mediastinum, urethra, bronchogenic,bladder, esophagus and colon, and Kaposi's Sarcoma, which is a form ofcancer associated with HIV-infected patients with Acquired ImmuneDeficiency Syndrome (AIDS). The composition may also be used for otheranti-proliferative conditions, such as arthrosclerosis and viralinfections, in particular AIDS. It may also be used in the treatment ofautoimmune diseases, including multiple sclerosis, rheumatoid arthritis,systemic lupus erythematosus, Type I diabetes, myasthenia gravis,Addison's Disease, autoimmune hemolytic anaemia, Crohn's disease,Goodpasture's syndrome, Graves' disease, Hashimoto's thyroiditis,idiopathic thrombocytopenic purpura, pernicious anaemia,poststreptococcal glomerulonephritis, psoriasis, scleroderma, Sjogren'ssyndrome, spontaneous infertility, and pemphigus vulgaris.

The compositions of the invention may be converted using customarymethods into pharmaceutical agents. The pharmaceutical agents containthe composition of the invention either alone or together with otheractive substances. Such pharmaceutical agents can be for oral, topical,rectal, parenteral, local, inhalant, or intracerebral use. They aretherefore in solid or semisolid form, for example pills, tablets,creams, gelatin capsules, capsules, suppositories, soft gelatincapsules, gels, membranes, and tubelets. For parenteral andintracerebral uses, those forms for intramuscular or subcutaneousadministration can be used, or forms for infusion or intravenous orintracerebral injection can be used, and can therefore be prepared assolutions of the compositions or as powders of the active compositionsto be mixed with one or more pharmaceutically acceptable excipients ordiluents, suitable for the aforesaid uses and with an osmolarity whichis compatible with the physiological fluids. For local use, thosepreparations in the form of creams or ointments for topical use or inthe form of sprays may be considered; for inhalant uses, preparations inthe form of sprays, for example nose sprays, may be considered.Preferably, the composition is administered intramuscularly.

The pharmaceutical compositions can be prepared by per se known methodsfor the preparation of pharmaceutically acceptable compositions whichcan be administered to patients, and such that an effective quantity ofthe active substance is combined in a mixture with a pharmaceuticallyacceptable vehicle. Suitable vehicles are described, for example, inRemington's Pharmaceutical Science (Nack Publishing Company, Easton,Pa., U.S.A. 1985).

On this basis, the pharmaceutical agents include, albeit notexclusively, the composition of the invention in association with one ormore pharmaceutically acceptable vehicles or diluents, and are containedin buffered solutions with a suitable pH and iso-osmotic with thephysiological fluids.

The compositions are indicated as therapeutic agents either alone or inconjunction with other therapeutic agents or other forms of treatment.For example, in the case of a malignant tumor, the present treatment mayrender a tumor suitable for surgical removal where it was not previouslyoperable. The compositions and agents of the invention are intended foradministration to humans or animals.

In general, a dosage range of the composition is envisaged foradministration in human medicine of from about 0.01 to 20 mg/kg,preferably from about 0.1 to 10 mg/kg, most preferably 0.1 to 1 mg/kg ofbody weight daily may be employed. In the case of intravenousadministration, the dosage is about 0.1 to 5 mg/kg of body weight daily,and in the case of oral administration the dosage is about 1 to 5 mg/kgof body weight daily. Where the concentrated composition is used,approximately half the above mentioned dosages may be used. For example,for intramuscular administration, a dosage of about 0.2 to 1.0 mg/kg ofbody weight daily, preferably 0.275-0.75 mg/kg of body weight daily maybe used.

It will be appreciated by medical practitioners that it may be necessaryto deviate from the amounts mentioned and, in particular, to do so as afunction of the body weight and condition of the animal to be treated,the particular disease to be treated, the nature of the administrationroute and the therapy desired. In addition, the type of animal and itsindividual behaviour towards the medicine or the nature of itsformulation and the time or interval at which it is administered mayalso indicate use of amounts different from those mentioned. Thus it maysuffice, in some cases, to manage with less than the above-mentionedminimum amounts whilst in other cases the upper limit mentioned must beexceeded. Where major amounts are administered, it may be advisable todivide these into several administrations over the course of the day.

Thus, the present invention comprises a process for preparing animmunomodulator composition comprising (a) mixing bile from an animalwith a water-soluble solvent to produce a bile/solvent solution; (b)isolating an aqueous solution substantially free of solvent from thebile/solvent solution; and (c) removing bile pigments from thesubstantially solvent-free solution to obtain a colorless liquid,preferably where the water soluble solvent is an alcohol, and where thebile from the animal is mixed with an equal volume of the alcohol.Preferred aspects of the aforementioned process also comprise furtherconcentrating the colorless liquid to about one-eighth, or one-tenth,the original volume of the bile/solvent solution. Obviously,compositions produced via the above process form a preferred aspect ofthe invention.

The present invention also comprises a composition for use as animmunomodulator, comprising at least one component having a molecularweight of less than about 3000 daltons, which shows no cytotoxicity tohuman peripheral blood mononuclear cells, and has at least one of thefollowing properties:

(a) is capable of stimulating monocytes and macro-phage in vitro or inviva to produce one or more cytokines;

(b) is capable of stimulating monocytes or macro-phages to produce tumornecrosis factor in vitro or in vivo; or

(c) has an anti-proliferative effect in a malignant mouse hybridoma cellline; and

wherein said component is not an endotoxin, IL-1α, IL-1β, TNF, IL-4,IL-6, IL-8, GM-CSF or IFN-gamma. Such compositions may be obtained fromthe bile of animals, preferably bovines, or from other sources. In apreferred embodiment of the composition, the composition stimulatestumor necrosis factor production in vitro or in vivo, and mostpreferably in humans, in the absence of exogenous IL-1α, IL-1β, TNF,IL-4, IL-6, IL-8, GM-CSF, and IFN-gamma,

The compositions of the present invention also have components which canbe characterized by column chromatography such that when saidcomposition is dried to obtain a solid residue, and 2 grams of saidresidue are dissolved in 20 ml of a 10% concentrated ammonium hydroxidesolution in methanol, and after any insoluble material is removed, issubjected to column chromatography in a methanol column havingdimensions of 5 cm×12.5 cm, and containing 102 g of 60 A flash silicagel, and operating at a pressure of 10 pounds per square inch and a flowrate of 11 mi/min with a 10% concentrated ammonium hydroxide in methanolsolvent solution, said component is eluted from the column in a fractiontaken when the total column elution is between about 180 and about 220ml, between about 220 ml to about 260 ml, or between about 260 ml andabout 300 ml.

Characterization of components may also be accomplished by ion-exchangechromatography, such that when 10 ml of said composition is subjected toanion-exchange chromatography in a column containing Bio-Rad AG-1hydroxide form resin in an amount sufficient to bind substantially allthe anions present in said 10 ml of said composition, said component iseluted from the column using a step gradient of ammonium bicarbonatebuffer at a buffer concentration from about 0.5 M to about 1.5 M,preferably at a buffer concentration from about 1.0 M to about 1.5 M,and most preferably at a buffer concentration of about 1.5 M.

Reversed-phase (C18) HPLC can also be used for characterization ofcomponents, such that when said composition is lyophilized andreconstituted in 0.1% TFA in water and then subjected to reversed-phase(C18) HPLC in a Phenomenex WP60009-C18 column, having dimensions of250×4.6 mm, where a first buffer of 0.1% TFA in water is run through thecolumn for about 10 minutes, then a linear gradient from 0 to 80% of asecond buffer of 0 1% TFA in acetonitrile is run for about 55 minutes,followed by an 80% solution of the second buffer for about 5 minutes,and an 80%-0% gradient of the second buffer for about 5 minutes, andwhere flow rate is 1 ml/min. and the capacity of the column and buffersare not exceeded, said component is eluted from the column at a timefrom about 2.4 minutes to about 3.4 minutes after said reconstitutedcomposition is applied to the column. Characterization of components ofthe composition can also be accomplished by an additional reversed-phaseHPLC method, such that when said composition is dialyzed or dissolved ina first buffer of 0.1% TFA in water and then subjected to reversed-phase(C18) HPLC in a Bio-Rad Hi-Pore RP 318 (C18) column, having dimensionsof 250×4.6 mm, where the first buffer is run through the column forabout 10 minutes, then a linear gradient from 0-80% of a second buffer,of 0.1% TVA in acetonitrile is run for about 55 minutesB followed by an80% solution of the second buffer for about 5 minutes, and an 80-0%gradient of the second buffer for about five minutes, and where the flowrate is 1 ml/min. and the capacity of the column and the buffers are notexceeded, said component is eluted from the column at a time from about2 minutes to about 21.4 minutes, or at a time from about 21.4 minutes toabout 25.6 minutes after said dialyzed composition is applied to thecolumn.

The compositions of the present invention can also be characterized byTLC, such that when said composition is subjected to thin layerchromatography on silica gel plates in 10% concentrated ammoniumhydroxide in methanol and visualized with a ninhydrin spray, a positivereaction with ninhydrin occurs at an R_(f) value from about 0.80 toabout 0.90.

The present invention also comprises a method of stimulating tumornecrosis factor production in humans, comprising administering aneffective amount of a composition comprising at least one of thefollowing compounds:

(a) a compound of the formula

where the bonds between A—B, B—C, and C—D may be single or double bonds,and where X═OH, ═O, or OSO₃H; and Y═

where R is an amino acid residue;

(b) a compound of the formula (R¹O)CH₂CH(OR²)CH₂(OR³—X) or

where R¹, R² and R³ are H, COR⁴, CH═CH—R⁵, X, P(O) (OH)O—, or —S(O)₂O—;

X is choline, ethanolamine, N-alkylated ethanolamines, serine, inotitol,sugars bearing free hydroxyls, amino-sugars, sulfonated sugars, orsialic acids; and

R⁴ is a saturated or unsaturated alkyl group having a carbon chain fromabout C₁ to C₃₀, or oxidized and hydroxylated analogs thereof; and

R⁵ is an alkyl group or oxidized and hydroxylated analogs thereof,

(c) a mucin hydrolysis product or a proteoglycan hydrolysis product; or

(d) a fat-soluble vitamin.

Preferably, compositions of the inventive method comprise at least onecompound selected from the group consisting of taurocholic acid and itssulphated derivatives; glycocholic acid and its sulphated derivatives;sphingosine; a diacyl glycerol; lecithin; an oligosaccharide of lessthan 10 saccharide units in length, where said oligosaccharide iscomprised of sialic acid, fucose, hexosamines, or sulphated hexosamines;Vitamin A; retinolic acid derivatives; retinol derivatives; taurine; andglutamic acid and its conjugates. The composition may also additionallycomprise at least one compound selected from the group consisting ofammonia; primary alkyl amines; secondary alkyl amines; tertiary alkylamines; and a carboxylic acid R⁶CO₂H, wherein R⁶ is C₁-C₃₀ alkyl that issaturated or unsaturated, and oxidized and/or hydroxylized derivativesthereof.

The method of the invention also embraces stimulation of TNF productionby administration of a composition comprising at least one compoundselected from the group consisting of taurocholic acid and its sulphatedderivatives; glycocholic acid and its sulphated derivatives;sphingosine; a diacyl glycerol; lecithin; an oligosaccharide of lessthan 10 saccharide units in length, where said oligosaccharide iscomprised of sialic acid, fucose, hexosamines, or sulphated hexosamines;vitamin A; retinoic acid derivatives; retinol derivatives; taurine;and-glutamic acid and its conjugates.

The present invention also provides a method of treating pancreaticcancer comprising administering to a patient suffering from said cancera therapeutically effective amount of the compositions of the invention.

Also forming part of the present invention are compositions comprising(1) micelles of sphingosine or sphingosine complexed with a salt, or (2)micelles of retinolic acid or its derivaties, which have at least one ofthe following properties:

(a) is capable Of stimulating monocytes and macrophages in vitro toproduce one or more cytokines;

(b) is capable of stimulating monocytes or macrophages to produce tumornecrosis factor in vitro or in vivo; or

(c) has an anti-proliferative effect in a malignant mouse hybridoma cellline.

The micelles may also comprise a diacyl glyceride or lecithin, and mayfurther comprise a bile acid salt, and a source of ammonium or alkylammonium ions.

Finally, the present invention also contemplates compositions comprising(1) sphingosine, a bile acid salt, and a source of ammonium or alkylammonium ions, (2) a bile acid salt, sphingosine, a diacyl glycerol, asource of ammonium or alkyl ammonium ions, and a retinal derivative, (3)a diacyl glyceride, lecithin, and a bile acid salt, or (4) (a) a diacylglyceride, (b) lecithin, and (c) a mucin hydrolysis product or aproteoglycan hydrolysis product, which has at least one of the followingproperties:

(a) is capable of stimulating monocytes and macrophages in vitro toproduce one or more cytokines;

(b) is capable of stimulating monocytes or macrophages to produce tumornecrosis factor in vitro or in viva; or

(c) has an anti-proliferative effect in a malignant mouse hybridoma cellline.

The following non-limiting examples are illustrative of the presentinvention:

EXAMPLE 1

Preparation of the Composition of the Invention

Bovine bile is collected from healthy herds at least one and one halfyears old which have been slaughtered for food use at a licensed andinspected abattoir. The gall bladders are collected from the slaughteredanimals which have been inspected and the gall bladders are separatedfrom the livers and examined by a veterinarian to confirm that the gallbladders are free of parasites and evidence of infection, and thus aresuitable for use as a source of bile.

Gall bladders which pass this inspection are wiped with a solution of70% ethanol to sanitize the exterior and a syringe is inserted to removethe bile. The bile removed is visually examined by the veterinarian inthe syringe to assure that it contains no blood or pus and is otherwisesatisfactory. Bile found to be satisfactory is transferred into agraduated amber bottle containing ethyl alcohol. The bile is a greenishfluid substantially free of blood and pus. Bile is added to each bottleto a level marked on the bottle, twice the level of ethanol present togive a 50% bile/ethanol solution. The bile/ethanol solution is agreenish fluid substantially free of foreign material in an approximate50%/50% bile/ethyl solution. It also shows positive for ethyl alcoholUSP XXII Part B. These bottles are labelled with the date of collectionwhich serves as the lot number. A minimum of fifty animals serve as thepool for each lot. Fragments of livers, spleen, and lymph nodes are alsocollected from the animals whose bile made up the pool and the fragmentsare examined for the presence of parasites or other indications ofdisease.

The bile/alcohol mixture is then centrifuged at 4200 RPM for at least 2½hours at 20+/−2° C. The supernatant liquid is decanted and checked forpH and ethanol content. The decanted liquid is then subjected to anactivated charcoal treatment. The treated bile/ethanol is then monitoredfor Optical Density (“O.D.”) and conductivity. O.D. levels orconductivity levels outside acceptable specifications will require thatthe bile ethanol solution be given additional treatment with activatedcarbon to achieve a reading within specification limits.

Following activated carbon treatment, the solution is filtered through afilter (for example using filters having a 2.5 μm retention), thealcohol is evaporated off (for example, by heating to less than 85° C.)and the solution is concentrated to approximately one eighth of theoriginal bile/ethanol solution volume. The concentrated solution iscooled to 20-25° C. This solution is then mixed with ethyl ether and theether phase is discarded. This step may be repeated once. The aqueousphase is heated to remove residual ether (for example by heating up toabout 55° C. for about 10 hours) and further reduced in volume to onetenth of the original bile/ethanol volume by heating to about 80-85° C.The resultant composition is then tested for appearance, biologicalactivity and ethanol and ether content. The composition is a clearyellowish solution essentially free of foreign matter, and it containsnot more than 10 ppm ethanol and not more than 5 ppm ether. In thebioassay described in Example 4, the non-proliferative growth is18+/−Units/ml.

Identity and purity are determined using reverse-phase high pressureliquid chromatography. Potency is assayed using the antiproliferativemethod as described in Example 4.

Initial batches of the composition of the invention were manufactured asa non-buffered liquid. Subsequent batches were manufactured as abuffered liquid, prepared by adjusting the pH of the composition toabout 7.4+/−0.05, using hydrochloric acid (1%) solution and sodiumhydroxide (1% solution), as well as using dibasic and monobasic sodiumphosphate salts as buffers. Bioburden reduction is conducted in a steamautoclave at 104+/−2° C. for 60 minutes. The bulk solution is filledinto 5 ml or 10 ml sterile bottles and capped. The filled and cappedbottles are subjected to three sterilization cycles by autoclaving themat 104° C.+/−2° C. for 60 minutes followed by incubation at 35° C. for23+/−1 hrs. Between each cycle (autoclave plus incubation samples) aretaken and tested for bioburden. Following the last cycle, the bottlesare visually inspected against a black and a white background to detectany particulates which may be present.

Following inspection, the lot is sampled and tested for conformance tospecifications. Tests include identity, sterility, pyrogenicity,endotoxin, bioassay, HPLC and general safety (See Table 1).

TABLE 1 Results for Individual Batches FINAL BATCH # BATCH # BATCH #PRODUCT TEST BC0226 BC0227 BC0228 Biological Activity 17 14.0 22.5 (18.0+/− 5 units/ml) Identity/Purity Pass Pass Pass Agrees with referenceSafety (Passes test) Pass Pass Pass Pyrogeniticy (temp. increase shallnot exceed 0.4° C.) Endotoxin ≦0.4 EU/ml ≦0.25 ≦0.25 ≦0.25 Sterility (nogrowth) Pass Pass Pass pH (7.40 +/− 0.05) 7.45 7.39 7.36 Appearance -Visual (clear, Pass Pass Pass light yellowish liquid with little or noprecipitate) Appearance - O.D (passes test) 0.088 0.118 0.088 Osmolarity540  603  445  IN-PROCESS TEST Solids (18 +/− 3 mg/ml) 18 15 20 AminoAcids 790  742  878  (800 +/− 10% mg/ml) Ethyl Alcohol Pass Pass Pass(not more than 10 ppm) Ethyl Ether Pass Pass Pass (not more than 10 ppm)Conductivity 25 22 29 (26 +/− 5 mMHO)

EXAMPLE 2

Physical Chemical and Biochemical Characteristics of the Composition ofthe Invention

A number of the physicochemical characteristics of the preparation(conductivity, osmolarity and total solids) are shown in Table 2. Moreparticularly, test results for three manufactured batches of acomposition prepared in accordance with Example 1 were carried out. Theresults shown in Table 2 demonstrate the sterility, potency, andreproducibility of the manufactured product. It is noted that the ethylalcohol and ethyl ether are measured as in-process tests only. A summaryof the method for determination of the biological activity is providedin Example 4.

TABLE 2 Product Specification Test Specification Method BiologicalActivity 18 +/− 5 units/ml Biological Activity Identity/Purity Agreeswith reference HPLC Safety Passes test General safety test (mice andguinea pigs) 21 CFR part 610.11 Pyrogenicity Temperature increasePyrogen test (rabbits) shall not USP exceed 0.4° C. Endotoxin <2 EU/mlLimulus Amoebocyte Lysate Test USP Sterility No growth Sterility TestUSP pH 7.40 +/− .05 pH test USP Appearance Clear, light yellowish VisualInspection liquid with little or no precipitate Solids 18 +/− 3 mg/mlLyophilization Amino Acids 800 +/− 10% ug/ml Trinitrobenzene- sulfonicAcid Method Osmolarity Freezing point depression USP Ethyl Alcohol Notmore than Direct Injection Gas 100 ppm Chromatography Ethyl Ether Notmore than Direct Injection Gas 100 ppm Chromatography Conductivity 26+/− 5 mMHO Copenhagen Radiometer Model

Physical and chemical properties such as conductivity, osmolarity andtotal solids are consistent with a composition of over 99% salt. Lessthan 1% of the solids in the composition is organic material, there areno lipids, around half are carbohydrates and the rest are amino acids.Proteins and peptides are present. SDS Gel electrophoresis confirmedthat there may be more peptides than proteins in the composition. Highmolecular weights are not detected. This is an important feature of apeptide drug because it is not expected to be immunogenic.

HPLC and bioassay test methods for the composition of the invention weredeveloped using the nonbuffered product. These tests are used tocharacterize the product as the buffered liquid and the concentratedformula. The HPLC results described below indicate that the product isthe same in all of the presentations. The bioassay shows that theactivity of the concentrated composition is two and a half times greaterthan the original composition. Therefore, the product used in thestudies has been demonstrated as being equivalent.

Reversed Phase (C₁₈) HPLC Analyses of the Composition of the Invention

The composition of the invention has a consistently reproducible patternon reversed phase HPLC in which peaks are seen early in the exclusionfraction and at about 27 and 32 minutes. Before, in-between and afterthe tall peaks, there are smaller peaks that vary in intensity. The HPLCreadings for three lots of the concentrated composition of the inventionare shown in FIGS. 1 to 3. RP-HPLC profiles for batches B0211 (FIG. 4),B0209 (FIG. 5), B29/3006 (FIG. 6) and B15/1606 (FIG. 7), also show avery reproducible pattern.

The RP-HPLC to characterize the composition of the invention was carriedout as follows. Bio-Rad Hi-Pore RP 318 guard column (C₁₈), 4.6×30 mm(Bio-Rad) and Bio-Rad Hi-Pore RP 318 (C₁₈) column, 4.6×250 mm was used.The samples were dialyzed in 0.1% trifluoroacetic acid (TFA Pierce) inH₂O (Buffer A) and applied to the column. Buffer A was run for 10minutes, then a linear gradient 0-80% of Buffer B (0.1% TFA in 100%acetonitrile) was run for 55 minutes. At the end of this period, 80%Buffer B was run for 5 minutes and 80-0% of Buffer B for 5 minutes. Flowrate was 1.0 ml/minute. Fractions from successive runs were collectedand pooled and concentrated in a Speedvac (Model SVC 200H, SavantInstruments, Farmington, N.Y.).

Preliminary Characterization of the Composition

Peaks from HPLC were submitted for protein sequencing. The initialsample, the major HPLC peak designated RP-HPLC −31.00 min, batch 0210 inTFA/CH₃CN, failed to yield data when subjected to N-terminal sequenceanalysis. The results may be interpreted as either a quantity problem orN-terminal blockage. Quantitation of protein content of that fraction byamino acid analysis after acid hydrolysis revealed that sufficientquantity should have been subjected to sequence analysis; however,because of the composition it was thought the sample may not be aprotein and thus N-terminal blockage would not be the problem. Thesamples displayed the following composition: about 70% Glx(glutamate/glutamine) plus about 15% glycinen (Gly). Furthermore,analysis of the equivalent sample from RP-HPLC gave similar results: 68%Glx and 15% Gly (Table 3). Further characterizations of unfractionatedmaterial plus several other fractions revealed the following. Thestarting material (32 mg/ml) yielded a sequence signal indicating apolyglutamate peptide/protein, consistent with the amino acidcomposition data.

TABLE 3 Mole % Glx Gly - HPLC Fraction run R1 (see profile) FractionAnalysis # Mole % SUM # (07/11/92) Glx Gly Glx + Gly 1a 393 28 44 721b + c 394 35 37 72 2a 395 39 36 75 2b 396 35 43 78 2c 397 44 30 74 3a398 69 21 90 3b 399 70 10 80 3c 400 52 18 70 4 401 68 15 83 402 48 30 78

Analysis of several HPLC fractions (Table 3) revealed that all are veryrich in Glx (Glu/Gln) (28-70 mol %) and Gly (10-44 mol %). Eachfraction, however, displayed real differences in the relative amounts ofthe other amino acids.

EXAMPLE 3

Biological Activity of Fractions of the Composition

The biological activity of fractions of the composition have beeninvestigated. The biological activity of the composition is thought tobe attributable to small molecular weight components (m.w. less than3000 daltons). This was determined through an experiment in which fourfractions of the composition and unfractionated composition were testedfor biological activity. The first fraction contained proteins andpeptides with molecular weight less than 3000 daltons, while theremaining three fractions contained additional larger molecular weightproteins and polypeptides. All fractions contained additional largermolecular weight proteins and polypeptides. All fractions andunfractionated compositions demonstrated the same biological activity.Since all fractions were as effective as the unfractionated product, andsince the common denominator of all fractions was the presence of thesame concentration of molecules smaller than 3000 daltons, this led tothe conclusion that the biological activity of the composition is due tocomponents with molecular weights smaller than 3000 Daltons.

EXAMPLE 4

Effect on Malignant Cell Lines

The effect of the composition of the invention on the proliferation ofcultures of four malignant cell lines (Daudi-human lymphoma cells,ME-180 human cervical carcinoma cells, T-24-human bladder carcinomacells, and mouse hybridoma cells #6-1) was measured. The composition ofthe invention had an antiproliferative effect on the mouse hybridomacells. The studies suggested that the inhibitory effect of thecomposition in mouse hybridoma cells is antiproliferative rather thancytocidal.

A bioassay based on the reproducible antiproliferative effect of thecomposition of the invention was designed in a mouse hybridoma cellmodel to facilitate characterization of the composition. The bioassay iscarried out as follows. The osmolarity and pH of the composition areadjusted to match that of the cell culture medium in order to isolatethe composition's biological activity from its physical and chemicalproperties. Serial dilutions of isotonic composition from 1:5 to1:10,000 are-prepared in culture medium. Hybridoma cell samples arespecifically quantitated. Using a hemocytometer the cells in a 100 μlsample are counted. The cells are concentrated by centrifugation andthen cell concentration is adjusted to 1,000 cells/ml (twice the finaldesired concentration) by addition of appropriate volumes of freshmedia. Hybridoma cell suspensions (1 ml) mixed with correspondingdilutions of the composition (1 ml) are incubated in 24 well plates at37° C. in a humid atmosphere with CO₂ controlled at 6%. After 96 hours,each well is sampled at 100 μl×3 and placed in a 96 well plate. (A blankof 100 μl of medium without cells is included.) Cell density isdetermined using a PROMEGA CellTiter 96™ kit. Cell concentration ismeasured by reading absorbance at 595 nm (650 nm reference) and recordedby ELISA plate reader. For each assay, a standard curve of cellconcentration is prepared. Cultured cells in the log phase of growth aresampled, counted, concentrated and resuspended in serial dilutions. Eachdilution is sampled at 100 μl×3 and placed in a 96 well plate. Thestandard curve is constructed by plotting cell density versus “net” ODat 595 nm after subtraction of zero cell blanks and OD at 650 nm. Celldensity of unknown samples is determined by interpolation.

To calculate biological activity, cell density as a percentage of thecontrol (no composition) is plotted against final composition dilution(in log and linear scale), and a curve is fitted through the pointsutilizing the Spline curve fitting method. Then, the compositiondilution that corresponds to 50% inhibition of cell proliferation isdetermined manually and converted directly to UNITS of composition. Bydefinition, one unit of composition inhibits by 50% the proliferation of1 ml cell culture of cell line HYB #6-1, seeded at 500 cells/ml, after96 hours at 37° C. and 6%CO₂. The average activity of the composition ofthe invention in the bioassay is 18.24+/−1.82 Units/ml.

EXAMPLE 5

Effect on T and B Lymphocytes in Culture

The composition of the invention has been shown to be non-toxic tonormal T and B lymphocytes in culture. The growth of human lymphocyteswas examined under carefully controlled conditions in the presence andabsence of the composition. Standard concentrations of lymphocytes wereincubated in wells containing various concentrations of the composition.When normal T and B human lymphocytes were incubated with thecomposition in concentrations similar to those that are used clinically,there were no adverse effects as judged by Trypan Blue exclusion.

The effect of the composition on the survival of human peripheral bloodmononuclear cells (PBMN) was examined. In this experiment, PBMN wereincubated for 24 and 48 hrs in plastic microwell plates with variousvolumes of the composition and tissue culture medium. At the end of thisperiod, the number of surviving cells was estimated by trypan blue dyeexclusion. Table 4 shows that the number of surviving cells fell at 24and again at 48 hours; however, the number of surviving cells in thepresence or absence of the composition was not different. Moreover,increasing volumes of the composition had no effect on survival (Table4). Thus, the composition showed no cytotoxicity to human PBMN.

TABLE 4 Concentration of Viable PBMN After Incubation No. of Live PBMNper Well by Trypan Blue (×10⁶)¹ Zero After 24 hrs No. After 48 hrs No.time % viable % viable Patient S.Z. Concentration (μl/well)  0 0.70²0.23 (33) 0.10 (14) 25 0.43 (61) 0.15 (21) 50 0.10 (14) 0.23 (33) 100 0.15 (21) 0.18 (26) 200  0.48 (69) 0.23 (33) LPS (μg/well)  1 0.30 (43)0.28 (40) 10 0.25 (36) 0.13 (18) Patient E.S. Concentration (μl/well)  01.30² 0.70 (54) 0.33 (25) 25 0.65 (50) 0.15 (12) 50 0.68 (52) 0.38 (29)100  0.75 (58) 0.23 (18) 200  0.65 (50) 0.20 (15) LPS (μg/well)  1 0.60(46) 0.53 (41) 10 0.15 (12) 0.15 (12) ¹Approximately 1 × 10⁶ cellsplated/well in triplicate. ²Actual number of cells counted/well (×10⁶).

EXAMPLE 6

Cytokine Content of Composition

ELISA assays for TNF-α, IL-1, IL-2, IL-4, IL-6, IL-8, GM-CSF and IFNwere conducted on the composition of the present invention. It wasdetermined that the composition of the invention contained no measurablelevels of cytokines (TNF, IL-1 alpha, IL-1 beta, IL-4, IL-6, IL-8,GM-CSF and IFN gamma) (See Table 5).

TABLE 5 ELISA DETERMINATION OF CYTOKINES IN COMPOSITION Cytokine 50 μl100 μl TNF pg/ml <5 <5 Detection Limit: 5 pg/ml IL-1β pg/ml — 6.5Detection Limit: 4.3 pg/ml GM-CSF pg/ml <5 — IL-6 pg/ml <7 — DetectionLimit: 7 pg/ml IFNγ pg/ml <5 — Detection Limit: 5 pg/ml IL-1α pg/ml <50— Detection Limit: 50 pg/ml IL-4 pg/ml — <3 Detection Limit: 3 pg/mlIL-8 ng/ml <4.7 Detection Limit: 4.7 ng/ml

EXAMPLE 7

Physical, chemical, and biological properties, were determined for anumber of batches of the composition of the invention prepared inaccordance with the method as described in Example 1. In addition, thechemical composition of the batches was determined and an amino acidanalysis of the batches was conducted. The results are shown in Tables6-8.

TABLE 6 CHEMICAL COMPOSITION High M.W. Solids Amino Acids SugarsLipids >3 kD PROT Batch No. mg/ml μg/ml μg/ml μg/ml μg/ml B0201 15.34.59 40.85 ND < 0.5 NA B0202 15.7 13.16 54.95 ND < 0.5 NA B0203 15.072.67 25.5 ND < 0.5 NA B0208 7.8 4.53 30 ND < 0.5 ND < 1.0 B0209 8.52.27 24 ND < 0.5 ND < 1.0 B0211 5.6 1.47 19.2 ND < 0.5 ND < 1.0 B010632.2 1.16 32.6 ND < 0.5 ND < 1.0 B0706 32.7 I.42 26.2 ND < 0.5 ND < 1.0B1306 22.3 8.01 48 ND < 0.5 ND < 1.0 B2006 21.7 9.73 38.4 ND < 0.5 ND <1.0 B2306 28.5 16.35 42 ND < 0.5 ND < 1.0 PHYSICAL, CHEMICAL ANDBIOLOGICAL PROPERTIES Absorban. UV, Batch Conduct. Osmolar. O.D. VISActivity No. pH mMHO mOsM 280 nm Peaks Units/ml B0201 7.37 16.9 361 0.98404 nm 10.5 B0202 7.35 17.3 298 0.777 None 6.5 B0203 7.3  17.7 360 0.67365 nm 21.0 B0208 7.00 16.1 250 0.453 None 8.1 B0209 7.31 11.2 259 0.594None 6.7 B0211 7.35 34.9 175 0.287 None 7.5 B0106 7.57 34.3 627 0.341None 17.2 B0706 7.57 11.6 627 0.387 None 23.0 B1306 8.02 35.6 790 1.147None 17.0 B2006 8.56 33.9 651 1.024 None 21.0 B2306 8.01 35.1 623 1.054None 19.0 Comments: 1. To batches No. B0106 and B0706 full isotonic PBSsolids were added. 2. Batches B1306, B2006 & B2306 were concentrated X2,no pH adjustment.

TABLE 7 CHEMICAL COMPOSITION High M.W. Solids Amino Acids SugarsLipids >3.5 kD PROT Batch No. mg/ml μg/ml μg/ml μg/ml μg/ml B0213 31.621 61 ND ND R0201/−pH 52.5 1553  216  ND ND R0201/+pH 55.8 1530  280  NDND C0203 36.1 113  42 ND ND 0-13/2109 12.1 149  36 ND ND B27/2806 17.528 37 ND ND B29/3006 28.7 26 60 ND ND B15/1606 26.8 41 45 75 NDPHYSICAL, CHEMICAL AND BIOLOGICAL PROPERTIES Batch Conduct. Osmolar,Absorban. UV, VIS Activity No. pH mMHO mOsM O.D. 280 nm Peaks Units/mlB0213 7.75 29.5 628 0.48 none 14.5 R0201−pH 7.95 44.5 877 1.59 271 nm51.5 0.65 O.D. R0201/+pH 7.60 50.0 1162  2.29 266 nm 61.5 1.6 O.D. C0203 7.90 34.8 657 0.96 NONE  5.0 0-13/2109 7.73 17.0 316 0.83 none14.5 B27/2806 7.71 22.0 453 0.49 none 12.4 B29/3006 7.67 28.8 605 0.55none 14.0 B15/1606 7.84 35.0 753 1.04 none 14.0

TABLE 8 AMINO-ACID COMPOSITION Batch # B-0208 B-0209 B-0211 01/06 07/061306 2006 2306 Asparagine 365 113 289 Serine 69 12 7 17 144 119 308Glycine 22 449 274 279 417 3731 5314 10371 Histidine 192 90 68 938 13352114 Arginine 161 533 Threonine 19 13 30 148 142 250 Alanine 173 112 2464 949 1002 1423 Proline 1092 74 817 639 1075 Tyrosine 15 55 57 43 39205 135 45 Valine 121 63 31 10 15 367 335 224 Methionine 970 461 462 13107 121 70 Cysteine 103 90 41 12 86 49 10 Isoleucine 2721 84 95 17 232216 68 Leucine 58 9 221 242 84 Phenylalanine 57 200 16 45 80 23 Lysine191 36 123 6 18 15 Total AA 4.53 2.27 1.47 1.16 1.42 8.01 9.73 16.35μg/ml

EXAMPLE 8

Activation of Monocytes and Macrophages

Investigations have shown that the composition of the invention willactivate normal monocytes to demonstrate cytotoxicity towards the Changhepatoma cell line which is used to measure monocyte toxicity and thatthe monocytes and macrophages from cancer patients (cervical and ovariancancer) have been stimulated by the composition to attack and destroytheir own particular tumor cells.

More particularly, the monocyte tumoricidal function has been tested inthe presence of the composition of the invention and the basic procedurefor these experiments is outlined below.

Venous blood is collected in heparinized vacutainer tubes. The blood isdiluted 3:1 in Hanks balanced salt solution (HBSS) layered ontolymphocyte separation medium and centrifuged to obtain a band ofperipheral blood mononuclear cells (PBMN). After centrifugation, themononuclear cell layer is recovered from the interface washed twice inmedium and monocytes are enumerated by latex ingestion. Nonocytes areisolated by adherence in 96 well plates (for 2 hours at 37° C. followedby two cycles of washing). Adherent cells are estimated to be greaterthan 90% monocytes. Wells containing adherent cells are incubatedovernight in the presence of the composition 11:10 dilution)granulocytemacrophage stimulation factor or PHA. Then adherent cells arewashed and incubated overnight with tumor cells. For studies using astandard cell line ⁵¹CR-labelled Chang hepatoma cells are used becausethis cell line is insensitive to natural killer cell cytotoxicity. Thesehepatoma target tumor cells are added to adherent cell monolayers at aneffector:target (E:T) cell ratio of 20:1. This E:T ratio is used becauseit falls well into the plateau range on a curve prepared by varying theE:T ratio from 5:1 to 30:1.) After 24 hours supernatants are collectedand ⁵¹Cr release is quantitated. The percent specific cytotoxicity iscalculated as:

% specific release=(E−S)/(T−S)×100

where E=CPM released from target cells in the presence of effectorcells; S=CPM released from target cells in the absence of effectorcells; T=CPM released from target cells after treatment with 2% sodiumdodecyl sulfate.

Using this protocol, the composition was found to cause monocytes fromhealthy donors to exert cytotoxicity toward the Chang hepatoma cellline. Subsequently, whether monocytes and macrophages from a cancerpatient could be stimulated by the composition to attack and destroytheir own particular tumor was investigated. Using similar protocols asdescribed for the standard cell line (Chang hepatoma cells), monocytesand/or peritoneal macrophages from cancer patients were isolated.(Peritoneal macrophages were isolated from peritoneal fluids collectedat the time of laparoscopy). The composition was found to activateperipheral monocytes and peritoneal macrophages from a patient withcervical cancer to produce cytotoxicity against the patient's own tumorcells. This effect was comparable to or better than that produced byinterferon or lipopolysaccharide. Peritoneal macrophages from a patientwith ovarian cancer were also found to be stimulated by the compositionto attack and destroy the ovarian tumor cells in culture.

EXAMPLE 9

Effect on TNF

Studies were conducted to evaluate the effect of the composition of theinvention on cytokine release from peripheral blood mononuclear cells(PBMN). ELISA assays for TNF-α, IL-1a, IL-2, IL-4, IL-6, IL-8, GM-CSFand IFN were conducted.

The following methods were used in the studies described in the Example.

In the studies of TNF, whole blood was drawn from 5 healthy subjectsinto heparinized Vacutainer tubes. Peripheral blood mononuclear cells(PBMNs) were isolated by gradient centrifugation on Ficoll-Hypaque(Pharmacia). The PBMN were washed twice with phosphate buffered saline(PBS), counted and resuspended in RPMI 1640 culture medium (Gibco Labs)at a concentration of 10⁶ cells/0.5 ml. These cells were cultured in 24well, flat-bottomed tissue culture plates (Falcon, Becton, Dickinson).Of the PBMN suspension, 0.5 ml was added to each well containing 50 ngLipopolysaccharide (LPS) (from E. coli), 10 μl fetal calf serum and therespective volumes of composition tested 10-300 μl). To neutralize thehyperosmolar effect of the composition, distilled water was added to theculture wells at a volume equivalent to 10% of the volume of compositionused. The total volume was then made up to 1 ml/well with RPMI. Ascontrol, PBS was used instead of composition. The cells were culturedfor 2, 6, 24, 48 and 72 hours at 37° C. in a humidified 5% CO₂incubator. At the end of each incubation period, the cells wereharvested and cell free culture fluids were obtained by centrifugationat 9000 rpm for 10 minutes. The samples were then stored at −70° C.until ELISA for cytokines was carried out (within 2 weeks).

Protein estimation of the composition was done using the Pierce MicroBCA Protein determination technique (Smith et al., Anal. Biochem. 1985,150:76-85). 10 μl of a sample of the composition was made up to 1 mlwith distilled water. Five concentrations of Bovine Serum Albumin (0.150μg/ml) was also made up to be used as standards. As a blank, 0.1N NaOHwas used. To all these samples was added a mixture of BCA, 2%Bicinchonic Acid sodium salt) (Pierce), Copper Sulfate 4% andMicroreagent A (NaCO₃, NaHCO₃, Na tartrate in 0.2N NaOH). The samplemixtures were incubated for 1 hr at 60° C., cooled and the resultantabsorbency read at 562 nm using a spectrophotometer. The amount ofprotein in the test sample was then compared to the plotted standardcurve and the appropriate calculations made. The protein concentrationof the composition was found to be low and estimated to be 32 μg/ml.

Cytokine synthesis in the supernatants were measured after stimulatinghuman PBMN with the composition of the invention at volumes of 200 and300 μl/well. The initial preparations of the composition show nostimulatory effect on cytokine production (Table 9). If there was anyeffect there was the suggestion that cytokine production was below theconstitutive level when PBMN were incubated in medium alone.

TABLE 9 Direct Effect of Composition on Cytokine Production after 24 hrsAmount of Cytokine Released (pg/ml)¹ Cytokine Composition LPS AssayedMedium 100 μl 200 μl 1 μg IL-1α 61.6 ± 12   59.6 ± 7.8  54.3 ± 6.0  315± 117 IL-1β 199 ± 184 218 ± 165 188 ± 174 965 ± 99  TNF² 203 ± 149 151 ±117 107 ± 120 1501 ± 284  IL-6 928 ± 776 853 ± 673 829 ± 543 2016 ± 41 IL-8 126 ± 70³   94 ± 50³  77 ± 41³  361 ± 165³ GM-CSF 13 ± 4  13 ± 7 15 ± 11 54 ± 20 IFN-γ 11 ± 18  9 ± 14 5 ± 6 54 ± 94 IL-4 <3.0 <3.0 <3.0<3.0 ¹Mean of eight patient samples in duplicate ²Mean of seven patientsamples in duplicate ³ng/ml

Experiments were performed to determine whether the composition of theinvention would impair LPS-stimulated release. LPS was used as apositive stimulus, and the ability of the composition to impair LPSstimulated release of cytokines was compared for the different cytokines(Table 10). The composition clearly inhibits IL-1 alpha, IL-1 beta andTNF. The effects on the other cytokines IL-6, IL-8, IFN-gamma and GM-CSFwere not as marked. However, in no instance did the compositions testedaugment the effect of LPS stimulated release of cytokines.

TABLE 10 Difference between LPS Released Cytokine and LPS plusComposition Stimulant of Cytokine Cytokine LPS LPS + Mean Assayed¹ (50ng/ml Composition² Difference IL-1α 129 ± 135 77 ± 27  −52 IL-16 1314 ±723  919 ± 460 −395 TNF 915 ± 763 497 ± 525 −418 IL-6 2320 ± 1081 2320 ±1145   0 IL-8 118 ± 62  109 ± 56   −9 (ng/ml) IFN-γ 30 ± 24 13 ± 10  −17GM-CSF 54 ± 65 50 ± 63  −4 ¹PBMN from six patients were tested induplicate ²Composition batch B0209

The effect of different volumes of the compositions of the invention ininhibiting LPS stimulated release of TNF was examined. FIG. 8 shows adose-response curve of the composition inhibiting release of TNF by PBMNstimulated with LPS. Ten μl of the composition inhibited about 10% andincreased close to 30% inhibition at 100 μl of the composition. Anotherbatch (B0201) of the composition inhibited LPS-induced TNF production at200, 100 and 10 μl by 45, 21 and 12 percent, respectively.

Similarly, IL-1 beta production was inhibited in a dose-dependent mannerby the composition: 100, 25 and 10 μl of the composition inhibited by16, 10 and 9 percent, respectively.

Different batches of the composition were examined for their effect onLPS-induced release of TNF. In summary, it was found that batches of thecomposition produced in the same way and from the same animal induced anidentical effect. However, changes in the method of preparation or thecomposition from different animal species had different effects. BatchesB29/3006, B0213 (B=bovine) and C0203 (goat) induced a strong release ofTNF above that induced by LPS alone (Table 11).

TABLE 11 Dose-Response Effect on TNF Release of Strong StimulatoryBatches of Composition Difference in TNFα Release Between LPS +Composition − LPS Alone Composition Batch Volume (μl) TNF (pg/ml) B0213 10 193 ± 161 100 858 ± 819 200 2131 ± 1742 B29/3006  10 121 ± 102  50422 ± 78  100 834 ± 811 200 2252 ± 676  C0203  10 101 ± 47   50 643 ±231 100 2650 ± 1372 200 1851 ± 980 

Table 12 shows the results with batches B15/1606 (concentratedpreparation) and B27/2806, which were moderately stimulatory.

TABLE 12 Dose Response Effect on TNF Release of Moderate StimulatoryBatches Difference in TNFα Release Between LPS + Composition − LPS AloneComposition Batch Volume (μl) TNF (pg/ml) B27/2806  10 −24 ± 120  50  71± 103 100 667 ± 844 200 984 ± 200 B15/1606  10 299 ± 351  50 294 ± 145100 667 ± 800 200 1224 ± 446 

Table 13 shows that batch 013/2109 (sheep) was minimally stimulatory.

TABLE 13 Dose-Response Effect on TNF Release of Minimal StimulatoryBatch Difference in TNFα Release Between LPS +   − LPS Alone BatchVolume (μl) TNF (pg/ml) 013/2109  50 −9 ± 73 200 179 ± 162 300 178 ± 373

Table 14 shows that batch R0201 (shark) was inhibitory at mostconcentrations for LPS-induced TNF production.

TABLE 14 Dose-Response Effect on TNF Release of Inhibitory BatchDifference in TNFα Release Between LPS +   − LPS Alone Batch Volume (μl)TNF (pg/ml) R0201  50 145 ± 256 200 −370 ± 385  300 −400 ± 185 

Initially, the composition was shown to affect LPS-induced release ofTNF from human PBMN. Thus, in the next series of experiments, the timeeffect of the composition on LPS-induced release of TNF was examined(Table 15). LPS stimulated release of TNF. By 2 hours the level hadrisen to 697 pg/ml and peaked at 6 hrs at about 2006 pg/ml. At 24, 48and 72 hrs the release of TNF progressively fell. In fact, by 48 and 72hrs, the TNF release was just above constitutive production levels. Bycontrast, Batch 0213 of the composition, which was strongly stimulatoryfor TNF release, showed no release of TNF above that produced by LPSalone at 2 and 6 hrs. Whereas LPS induced peak release of TNF at 6 hrs,the composition in combination with LPS induced peak release at 24 hrsat a time when the stimulatory effect of LPS had begun to fall. UnlikeLPS alone, composition+LPS continued to stimulate TNF release at 48 and72 hrs although the quantity of TNF released fell progressively (Table15). Thus batch 0213 was stimulatory for TNF release. Batch B15/1606,which was only moderately stimulatory, inhibited LPS-induced release at2 and 6 hrs. At 24 hrs, B15/1606 in combination with LPS was mildlystimulatory for TNF-release. At 48 and 72 hrs, B15/1606 in combinationwith LPS, had a mild stimulatory effect on TNF release. Thus, batchB15/1606 had a biphasic effect; early it inhibited LPS induced TNFrelease, and mainly at 24 hrs combination with LPS, had a mildstimulatory effect on TNF release. Thus, batch B15/1606 had a biphasiceffect; early it inhibited LPS induced TNF release, and mainly at 24 hrsit caused a mild additive effect in conjunction with LPS in inducingTNF-release.

TABLE 15 Time Effect on Different Batches on TNF Release by LPS MeanDifference in TNFα (pg/ml) Release Between LPS + Composition Time TNFReleased by LPS Alone by Three Different Batches (hr) LPS (50 ng/ml)BO213 B15/1606 R0201  2 697 ± 94  693 ± 339 363 ± 189 62 ± 42  6 2006 ±736  1949 ± 422  1080 ± 377  430 ± 260 24 800 ± 222 2301 ± 658  876 ±351 343 ± 183 48 170 ± 149 1419 ± 447  234 ± 183 129 ± 78  72 132 ± 147945 ± 367 184 ± 107 153 ± 68 

Batch R0201, which was inhibitory for LPS-induced release of TNF, wasmarkedly inhibitory for LPS induced TNF release at 2, 6 and 24 hrs. At48 and 72 hrs, LPS induced minimal TNF release and batch R0201 hadminimal positive or negative affects at these times.

The direct stimulatory effect of Batch B0203 was tested at differentvolumes and then at different times.

Batch B0203 stimulated maximum TNF-release at about 100 μl (Table 16).The maximum effect was observed at 24 hrs for a volume of 200 μl (Table17). Thus, the composition was able to stimulate TNF-release on its own,that is in the absence of LPS and the curves for TNF-release weresimilar to when the composition and LPS were combined. It appears thatthe composition by itself was less stipulatory for TNF release than theadditive effect it had when combined with LPS.

TABLE 16 Dose-Response of Batch B0203 on Stimulating Release of TNFVolume Mean Amount of (pg/ml) TNF Released at (μl) 24 hrs None  128 ±207 (PBS 50 μl)  50 223 ± 65 100 327 ± 90 200 105 ± 54 300 189 ± 94

TABLE 16 Dose-Response of Batch B0203 on Stimulating Release of TNFVolume Mean Amount of (pg/ml) TNF Released at (μl) 24 hrs None  128 ±207 (PBS 50 μl)  50 223 ± 65 100 327 ± 90 200 105 ± 54 300 189 ± 94

In summary, the experimental results indicate that some batches of thecomposition are able to inhibit TNF release when human PBMN-arestimulated with LPS. Other batches have biphasic effects suggesting thatthey partially inhibit LPS-induced TNF release and have, as a lateeffect, the ability to induce a mild release of TNF. A third preparationhad no inhibitory effect on LPS-induced release of TNF; but at adifferent time point than LPS, the preparation was able to stimulatehuman PBMN to release TNF. In conclusion, the composition of theinvention can modulate TNF production, an important mediator ofantitumor responses. A summary of the data is shown in FIG. 9 and inTable 18.

TABLE 18 TNF Bioassay Results Batch RP-HPLC Normal or TNF No. Peaks 1Min Source Concentrated Buffer Release B0213 27, 32 Bovine Normal Yes ↑↑ C0203 27, 32 Caprine Normal Yes ↑ ↑ 013/2109 27, 32, 21:50, OvineConcentrated Yes ↓ / ↑ 25 R0201 21-25, 28, 29, Shark Normal Yes ↓ ↓29.5, 27, 32 B29/3006 27, 32 Bovine Normal Yes ↑ ↑ B27/2806 ↓27, ↓32Bovine Normal Yes ↑ B15/1606 27, 32, 22, 28 Bovine Concentrated Yes ↑Yes ↓

EXAMPLE 10

This Example demonstrates, in summary, the following: The compositionhas TNF-α releasing activity and the TNF-α releasing activity is notrelated to any contamination with endotoxin. Priming of macrophagesenhances the ability of the composition to stimulate release of TNF-α.The hyperosmolarity of the composition is not responsible for TNF-αreleasing activity. The TNF-α releasing activity of the composition canbe separated, in part, from other constituents. The TNF-α releasingactivity of the composition does not bind or binds poorly to C₁₈RP-HPLC. Most of compositions activity elutes early from RP-HPLC. Lessthan 20% of the activity of the composition is recoverable from thefractions that are retained on the RP-HPLC and elute later. The TNF-αreleasing activity is precipitated by 80% acetonitrile, a high contentof organic buffer. The precipitated material when reconstituted inaqueous buffer and analyzed on RP-HPLC shows great similarity to theexcluded peak on RP-HPLC of the composition. It is possible to separateand concentrate the active component of the composition in a fractionthat constitutes about 30% of the original material.

A. Polymyzin and TNF-alpha Release

To eliminate any possibility of an endotoxin effect of the compositionexperiments were performed with Polymyxin added to the reactants.Polymyxin inhibits the action of endotoxin on leukocytes. Table 19 showsthat polymyxin completely inhibits the LPS-induced release of TNF-α. Inthe absence of polymyxin, LPS induces 517 pg/ml of TNF-α, whereas in thepresence of Polymyxin 11 pg/ml of TNF-α is released. The composition, onthe other hand, releases 1591 pg/ml of TNF-α in the presence ofPolymyxin. In the absence of Polymyxin, LPS and the composition showmore than just an additive effect of the stimulators, suggesting thatthe composition acts with greater intensity when macrophages are primed.

TABLE 19 Effect of Polymyxin on TNF Release by LPS + Composition SampleTNF Released (pg/ml) Tested Additive Total −LPS LPS Polymyxin 11 ± 7   0 None 517 ± 118   0 Composition Polymyxin 1591 ± 413  1581 (#B0213)None 5256 ± 2585 4738 Notes: 1. Total TNF Released is corrected for TNFrelease by 1640 Medium. 2. Polymyxin concentration: 50,000 units/ml. 3.Composition volume: 200 μl. 4. With polymyxin, 8 patients tested. Withno additive, 3 patients tested. 5. LPS concentration: 50 ng/10 μl.

B. TNF-Releasing Activity of Reversed-Phased High Pressure LicruidChromatoaraphy (RP-HPLC) Fractions

FIG. 10 shows the C₁₈ RP-HPLC profile of the comnposition, Batch 0213and the 5 separate fractions that were tested for TNF-releasingactivity.

Initially, the effect of different fractions were examined in thepresence of Polymyxin. Table 20 shows that the early eluting fraction(2:05 to 21:20 minutes) from RP-HPLC had most of the detectableTNF-releasing activity. However, this activity was only about 50% of thestarting composition. The right hand column of Table 20 shows theosmolarity of the samples. Batch B0213 was 369 and high. Fractions from21 minutes and later had normal osmolarity, whereas the first fractionwhich was active was even more hyperosmolar than the starting material,indicating that much of the salt in the composition also eluted early.Therefore, whether the hyperosmolarity of the samples was inhibiting orenhancing TNF-α release was investigated as set out below.

TABLE 20 Separation and Testing of Different HPLC Fractions ofComposition in the Presence of Polymyxin Sample TNF Released (pg/ml)Osmolarity Tested Total −LPS (mOsm) 1640 RPMI Medium 0 — 287 LPS (50ng/10 μl) 40 ± 25  0 Composition #B0213 1222 ± 448  1182  369 Fractions(minutes)  2:05-21:20 554 ± 394 514 434 21:20-25:32 7 ± 7  0 30125:32-31:55 0  0 299 31:55-34:58 4 ± 4  0 292 34:58-46:55 32 ± 28  0 295Note: 1. Total TNF Released is corrected for TNF release by RPMI Medium.2. Fractions are reconstituted in PBS. 3. Volume of composition andfractions: 200 μl. 4. Polymyxin concentration: 50,000 units/ml. 5.Number of patients tested: 5. 6. Osmolarity not measured for LPS.

Table 21 shows additional testing on two patients with compositionfractions 1 (2:05-21:20) and 2 (21:20-25.32). Once more, most of theactivity was recovered in fraction 1, but there was some activity infraction 2. However, fraction 1 and 2 had only about 50% of the startingactivity of the composition.

TABLE 21 Repeat Evaluation of Composition HPLC Fractions 2:05-21:20 and21:20-25:32 minutes for release of TNF in the Presence of Polymyxin.Sample TNF Released (pg/ml) Tested Total −LPS 1640 RPMI Medium 0 — LPS(50 ng/10 μl) 31 ± 31  0 Composition #B0213 2013 ± 726  1983  Fractions(minutes):  2:05-21:20 526 ± 126 496 21:20-25:32 132 ± 108 101 Note: 1.Total TNF Released is corrected for release by RPMI Medium. 2. Fractionsare reconstituted in PBS. 3. Volume of composition and fractions: 200μl. 4. Polymyxin concentration: 50.000 units/ml. 5. Number of patientstested: 2

To determine whether there was any nonspecific activity in thefractions, a blank run, was made and the same fractions were collected,concentrated and tested. The blank fractions produced virtually no TNFrelease. This result indicated that fractions from RP-HPLC could be usedto test for TNF-α releasing activity without concern that the column orbuffers contributed to TNF-α releasing activity.

Next the fractions of the composition were tested in the absence ofPolymyxin in order to have the priming effect of LPS. Table 22 showsthat Batch B0213 induced a marked release of TNF-α. There were 5fractions of the composition from the RP-HPLC, with the elution times asindicated. Once again, fraction 1 had the most TNF-α releasing activity.However, with the priming effect of LPS, fractions 2 through 4 had someTNF-α releasing activity. Fraction 2 (21:20-25:32) had about 25% of theactivity of fraction 1, and double the activity of the later fractions.

TABLE 22 Separation and Testing of HPLC Fractions of Composition in theAbsence of Polymyxin. Sample TNF Released (pg/ml) Osmolarity TestedTotal −LPS (mOsm) 1640 RPMI Medium 0 — 299 LPS (50 ng/10 μl) 219  0 305Composition #B0213 1575 ± 470  1356  376 Fractions (minutes): 2:05-21:20 656 ± 206 436 321 21:20-25:32 345 ± 82  126 309 25:32-31:55287 ± 70   68 305 31:55-34:58 262 ± 50   43 304 34:58-46:55 237 ± 59  18376 1. Total TNF Released is corrected for release by RPMI Medium. 2.Fraction Reconstitution: 2:05-21:20 in water, 21:20 to 48:55 in PBS. 3.Volume of composition and fractions: 200 μl. 4. Number of patientstested for TNF release: 5. 5. Osmolarities are averages for 2 of 5patients; standard errors very small, therefore, not reported.

Whereas Table 22 shows the results of using 200 μl volumes, Table 23shows the results of testing an additional three patients with 100 μl ofthe composition and 100 μl of its RP-HPLC fractions in the absence ofPolymyxin. Although the release by the composition is lower than with200 μl of the composition (Table 22), the results are similar. Fraction1 contains most of the activity with some activity in the laterfractions 2 and 3.

TABLE 23 Separation and Testing of HPLC Fractions of Composition in theAbsence of Polymyxin. Sample TNF Released (pg/ml) Osmolarity TestedTotal −LPS (mOsm) 1640 RPMI Medium 0 — 303 LPS (50 ng/10 μl) 195 ± 72  0 302 Composition #B0213 692 ± 266 497 347 Fractions (minutes): 2:05-21:20 575 ± 82  379 310 21:20-25:32 226 ± 65   31 337 25:32-31:55210 ± 71   14 305 31:55-34:58 192 ± 40   0 313 34:58-46:55 182 ± 73   0344 1. Total TNF Released is corrected for release by RPMI Medium. 2.Fraction Reconstitution: 2:05-21:20 in water, 21:20 to 48:55 in PBS. 3.Volume of composition and fractions: 200 μl. 4. Number of patientstested for TNF release: 3. 5. Osmolarities are averages of patientstested; standard errors very small, therefore, not reported.

C. Effect of Osmolarity of TNF Release by the Composition

The composition of the invention is hyperosmolar. The effect of thehyperosmolarity of the composition on TNF-α releasing activity wasstudied. It was found that the composition, when adjusted for osmolalityeven to the point of being hypoosmolar, continued to release TNF-α.

D. Physicochemical Separation of the Composition by Precipitation WithHigh Content of Organic Solvent

Since most of the TNF-releasing activity of the composition did not bindto the RP-HPLC as evidenced by its quick elution, it was decided to usea column that acts on the inverse principle of reversed-phasechromatography separation where the sample is in a high content oforganic solvent and permits hydrophilic interaction. This separationtechnique is used for small polar substances. However, when thecomposition was brought to 80% acetonitrile a precipitate formed. Thus,some of the contents of the composition in a high organic solvent bufferprecipitated. The precipitate and the soluble fraction were separated.Both the precipitate and soluble fraction were taken to dryness bylyophilization. The precipitate and soluble fraction were reconstitutedin aqueous solutions and both analyzed by RP-HPLC and tested for TNF-αreleasing activity. Table 24 shows that most of the TNF-α releasingactivity was contained in the precipitated material.

TABLE 24 TNF-Releasing Activity of Fractions of Composition Prepared byPrecipitation in 80% Acetonitrile. Sample Volume/ TNF Released (pg/ml)Osmolarity Tested Concentration Total −LPS (mOsm) 1X 199 Medium 0 — 306LPS 50 ng/ 161 ± 50   0 301 10 μl Composition 100 μl 471 ± 304 310 307#B0213 200 μl 505 ± 210 344 318 Supernatant 100 μl 192 ± 63   31 309 200μl 221 ± 69   60 310 Precipitate 100 μl 626 ± 212 465 307 200 μl 1299 ±565  1138  346 Note: 1. Total TNF Released is corrected for 1X 199Medium. 2. Reconstitution: supernatant in PBS, precipitate in doubledistilled water. 3. Precipitate is in (hypotonic) 70% 1X 199 Medium. 4.Number of patients tested: 5. 5. Osmolarities are averages of 4 of 5patients tested; standard errors very small, therefore, not reported.

RP-HPLc analy sis of both the precipitated (FIG. 11) and solublefractions (FIG. 12) of the composition shows that the precipitate isprincipally the material contained in fraction 1 of the RP-HPLC of thecomposition (FIG. 10), and the soluble material contains the otherfractions of the RP-HPLC of the composition (FIG. 10). Thus in twodifferent ways, it was shown that the composition's activity iscontained in the fraction that is minimally retained by RP-HPLC. Infact, the precipitate had equal activity to unprecipitated compositionwhen tested at 100 and 200 μl.

Only the 200 μl precipitate had above normal osmolarity. Consequently,the precipitate and soluble fractions (supernatant) of the compositionwere separated by RP-HPLC (FIGS. 11 and 12) and divided into twofractions (see profiles of RP-HPLC). Fraction 1 (2:00-21:10 min) wasequivalent to the same fraction 1 of the composition separated byRP-HPLC and fraction 2 was equivalent to fractions 2 through 5 of thecomposition separated by RP-HPLC. The isolates were then tested fortheir TNF-α releasing activity. Table 26 shows that for two patientsneither the precipitate or supernatant after RP-HPLC separation had anyTNF-releasing activity. However, the results for the initial twopatients (Table 25) suggested the possibility that the precipitate maynot have been tested at the ideal volume. Consequently, the fractionswere retested on two additional patients and at one lower concentration.Table 26 shows that at 50 and 100 μl, fraction 1 of the precipitate hadthe most activity. At 50 μl it released twice as much TNF-α as did 50 ngLPS. However, minor releasing activity was also found in RP-HPLCfraction 2 of the precipitate as well as minor activity was found onfractions 1 and 2 of RP-HPLC of the supernatant.

TABLE 25 TNF-Releasing Activity of HPLC Separated Fractions of 80%Acetonitrile Precipitated Composition. Osmol- Sample 199 Med- Volume/TNF Released (pg/ml) arity Tested ium Used Conc. Total −LPS (mOsm)Medium 1X 0 — 294 LPS 1X 50 ng/ 194 ± 93  0 294 10 μl (#B0213) 1X-70% 1X100 μl 855 ± 88  661  281 200 μl 926 ± 163 732  291 Supernatant 2:15-21:28 1X 100 μl 92 ± 75 0 269 1X 200 μl 25 ± 25 0 242 21:48-46:1370% 1X 100 μl 61 ± 56 0 296 70% 1X 200 μl 2 ± 2 0 297 Precipitate: 2:00-21:10 1X 100 μl 183 ± 15  0 305 1X 200 μl 0 0 354 21:10-46:20 70%1X 100 μl 62 ± 61 0 299 70% 1X 200 μl 0 0 302 Note: 1. Total TNFreleased is corrected for TNF release by 199 Medium 1X. 2.Reconstitution: first fractions in double distilled water, secondfractions in PBS. 3. Number of patients tested: 2. 4. Osmolarities areaverages of patients tested: standard errors very small, therefore notreported. 5. Averages of values in 1X and 70% 1X 199 Medium.

TABLE 26 TNF Release with further Titraton of Precipitate andSupernatant from 80% Acetonitrile Fractioned. Sample Volume/ TNFReleased (pg/ml) Osmolarity Tested Conc. Total −LPS (mOsm) 1X 199 Medium 0 — 317 LPS 50 ng/ 136 ± 38  0 320 10 μl #B0213 100 μl 274 ± 80 138 317 Supernatant  2:15-21:28 50 μl 171 ± 66 35 319 100 μl 193 ± 73 57 32221:48-46:13 50 μl 184 ± 34 48 311 100 μl 162 ± 40 26 310 Precipitate 2:00-21:10 50 μl 287 ± 69 150  333 100 μl 204 ± 40 68 355 21:10-46:2050 μl 148 ± 46 11 323 100 μl 198 ± 44 62 325 200 μl 1299 ± 565 1138  346Note: 1. Total TNF released is corrected for TNF release by 199 Medium1X. 2. Reconstitution: first fractions in double distilled water, secondfractions in PBS. 3. Number of patients tested: 2. 4. Osmolarities areaverages of patients tested: standard errors very small, therefore, notreported. 5. All samples in 1X 199 Medium.

E. TNF-α Releasing by Different Media

It was observed that the seitch from RPMI 1640 to Medium 199 resulted ina lower TNF-α release was evaluated in Medium 199 and RPMI 1640 (Table27). The results show that LPD from 10 to 200 ng is much more effectivein releasing TNF-α in RPMI 1640 Medium than in Medium 199. Presumablythe composition also gives greater release in RPMI 1640 Medium. Thus,cultural conditions can influence the degree of TNF-α release.

TABLE 27 Evaluation of TNF Release in Different Media 1X 199 Medium 1640RPMI Medium TNF TNF Sample Released Osmolarity Released OsmolarityTested (pg/ml) (mOsm) (pg/ml) (mOsm) Medium  23 296  47 LPS:  10 ng/10μl 124 291 —  50 ng/10 μl 155 292 356 285  100 ng/10 μl 147 293 323 289 200 ng/10 μl 213 294 455 288 1000 ng/10 μl 404 298 558 292 Note: 1.Number of patients tested: 1.

F. Osmolarity of the Composition

Table 28 shows the osmolarities of different batches of the composition.B0213 is moderately high at 675 mOsm. B0222 shown to have TNF-releasingactivity even better than B0213 is less hyperosmolar, 581 mOsm. Thefractions B0226, BC11-06 and BC11-09 range from 540 to 603 mOsm.

TABLE 28 OSMOLARITIES OF WHOLE BATCHES Osmolarity Batch # pH (mOsm)Concentrated: B0222 pre-pH 411 B0222 pH adjusted 581 B0216 pH adjusted872 B0219 pH adjusted 886 Nonconcentrated: B0221 pre-pH 652 B0221 pHadjusted 533 B0213 pH adjusted 675 B0225 pH adjusted 590 B0226 pHadjusted 540 BC 11-06 pH adjusted 445 BC 11-09 pH adjusted 603

EXAMPLE 11

A. Tumor Necrosis Factor (TNF) Releasing Activity of a Composition ofthe Invention

1. Acetonitrile Precipitate and Supernatant of the Composition

As, shown in the prior Example, 80% acetonitrile precipitated materialform a composition of the invention. The precipitated material and theunprecipitated (hence forth called Supernatant) composition were testedfurther to determine where the TNF-releasing activity resided.

Table 29 shows that the TNF-releasing component of the composition isprecipitated by 80% acetonitrile, an organic solvent. Whereas the wholecomposition at 0.04 ml released about 15 pg/ml of TNF, 0.05 ml ofprecipitated Batches of the composition released 58 (B0222), O (B0221),and 17 (B0213) pg/ml, suggesting recovery of the TNF-releasing componentby 80% acetonitrile precipitation. The precipitated composition wasreconstituted in the same volume of liquid from which it had beenprecipitated. Thus, 0.1 ml of precipitate comes from 0.1 ml of wholecomposition and equals 0.1 ml whole composition.

TABLE 29 TNF Releasing Activity of Precipitates of Virulizin* Preparedin 80% Acetonitrile. 199 Quantity Osmol- Sample Medium in TNF Released(pg/ml) arity Tested Used Wells Total −LPS (mOsm) LPS 1X  50 ng 322 ±115  0 308 Virulizin: B0222 Whole 80% 1X  40 μl 337 ± 107  15 312Precipitate 70% 1X 200 μl 1091 ± 137  769 351 70% 1X 100 μl 620 ± 186298 317 70% 1X  50 μl 380 ± 132  58 297 70% 1X  25 μl 312 ± 137  0 294Virulizin: B0221 Whole 90% 1X  40 μl 282 ± 75   0 306 Precipitate 70% 1X200 μl 981 ± 205 660 348 70% 1X 100 μl 526 ± 169 205 314 70% 1X  50 μl308 ± 104  0 298 70% 1X  25 μl 318 ± 185  0 292 Virulizin: B0213 Whole90% 1X  40 μl 383 ± 72   61 312 Precipitate 70% 1X 200 μl 1143 ± 172 821 366 70% 1X 100 μl 687 ± 186 365 326 70% 1X  50 μl 339 ± 133  17 30570% 1X  25 μl 300 ± 144  0 298 Note: 1. Number of patients tested: 5. 2.Total TNF released is corrected for TNF release by 1X 199 Medium. 3.Osmolarities not corrected for 1X 199 Medium (311 mOsm). 4. Average ofosmolarities given; standard errors not reported as values are very low.5. Precipitate reconstituted in double distilled water. 6. LPS volumeadded to wells was 10 μl. 7. Wells contained a total volume of 1000 μl.8. Sample volumes are equivalent. *Composition of the invention is alsoreferred to herein as VIRULIZIN

It is of interest to note that in earlier studies generally between 0.1and 0.2 ml of the composition was used to stimulate TNF-release. Theprecipitates of B0222, B0221 and B0213, reconstituted to 0.1 and 0.2 mlreleased between 205 and 821 pg/ml of TNF. At 0.1 ml of precipitate,batches B0222, B0221 and B0213 released between 205 and 365 pg/ml ofTNF, a very similar quantity of TNF, indicating the consistency of theTNF-releasing activity of the three different batches.

On a separate group of donor leukocytes, the supernatants remainingafter 80% acetonitrile precipitation of the composition were tested.Whereas 0.04 ml of whole batches of the composition (B0222 and B0213)released 175 and 233 pg/ml of TNF, 0.05 ml of the supernatant released42 and 41 pg/ml about 33% of the activity of the whole composition.Blanks prepared in the same fashion with 80% acetonitrile had noTNF-releasing activity. Thus, TNF-releasing activity in the precipitatewas not the result of some residual substances in the buffers used toinduce the precipitate.

Whereas in the aforedescribed studies the precipitate and supernatantfractions were tested on leukocytes from different donors, another studywas conducted in which the two fractions were tested on leukocytes fromthe same donors. For the two tested batches, the whole composition at0.04 ml released between 0 and 41 pg/ml of TNF. In comparison, theprecipitate of the same batches of composition at 0.05 ml released 141and 749 pg/ml of TNF, whereas 0.05 ml of the supernatant fractionreleased between 6 and 57 pg/ml of TNF. Thus the precipitate containedmuch of the TNF releasing activity. The supernatant fraction still hadsome TNF-releasing activity but much less than the precipitate and nomore than the whole composition.,

2. Reversed Phase-HPLC Separated Fractions of the Composition of theInvention

As the precipitate of the composition was shown to contain much of theTNF-releasing activity, the profile of the precipitate was examined byC₁₈RP-HPLC. FIGS. 18, 19 and 20 show the RP-HPLC profiles of wholecomposition, the precipitate and the supernatant, respectively. Theprecipitate's profile shows principally the early eluting peak of wholecomposition (FIG. 19), whereas the supernatant's profile (FIG. 20) issimilar to the whole composition except that the early peak is lessintense.

In the next series of experiments, the activity of the precipitate andsupernatants were evaluated after their separation by C₁₈ RP-HPLC. Theprecipitate and supernatant were collected as 2 pools from RP-HPLC: 2 to21 min and 21 to 46 min. Earlier studies of fractions of wholecomposition separated by RP-HPLC indicated that TNF-releasing activityeluted principally in the 2 to 20 min fraction. Table 30 shows theresults of testing whole composition and RP-HPLC fractions of theprecipitate and supernatant. In this particular experiment, only 10 μlof the whole composition was used and caused no TNF-release. Theprecipitate pool from 2 to 21 min released TNF, whereas the 21 to 46 minpool did not (Table 30). The supernatant pool from 2 to 21 min alsoreleased TNF activity, whereas the 21 to 46 min pool released minimalquantities of TNF (Table 30). Thus for both the precipitate andsupernatant, the TNF-releasing activity resides principally in the earlyeluting fraction from C₁₈ RP-HPLC.

TABLE 30 TNF Releasing Activity of HPLC Fractions of Precipitate andSupernatants of Virulizin Prepared in 80% Acetonitrile (2× wash) 199Medium Quantity in TNF Released (pg/ml) Osmolarity Sample Tested UsedWell Total −LPS (mOsm) LPS 1X 50 ng 99 ± 43 0 307 Virulizin: B0222 80%1X 10 μl 83 ± 33 0 289 Whole HPLC Precipitate:  2:00-21:36 70% 1X 100 μl153 ± 43  54  306 70% 1X 50 μl 50 ± 14 0 289 21:36-46:28 1X 100 μl 97 ±29 0 306 1X 50 μl 103 ± 43  3 306 HPLC Supernatant:  2:00-21:35 1X 100μl 192 ± 55  92  330 1X 50 μl 183 ± 72  83  320 21:35-46:30 1X 100 μl 65± 21 0 311 1X 50 μl 142 ± 40  43  309 Virulizin: B0213 90% 1X 10 μl 93 ±36 0 296 Whole HPLC Precipitate:  2:00-21:12 70% 1X 100 μl 165 ± 52  66 310 70% 1X 50 μl 48 ± 15 0 287 21:12-46:12 1X 100 μl 88 ± 31 0 307 1X 50μl 101 ± 38  1 307 HPLC Supernatant:  2:00-21:15 1X 100 μl 193 ± 76  93 317 1X 50 μl 126 ± 43 26  306 21:15-46:20 1X 100 μl 59 ± 24 0 309 1X 50μl 126 ± 32  26  312 Note: 1. Number of patients tested: 5. 2. Total TNFreleased is corrected for TNF release by 1X 199 Medium. 3. Osmolaritiesnot corrected for 1X 199 Medium (309 mOsm); standard errors are very lowand, therefore, not reported. 4. Reconstitution: precipates in Type 1water, supernatants in PBS buffer. 5. LPS volume added to wells was 10μl. 6. Wells contained a total volume of 1000 μl.

The results suggest that the TNF-releasing substance in the precipitateand supernatant are likely closely related molecules, if not identical,with the only difference, if any, perhaps being the degree of solubilityin 80% acetonitrile.

In another experiment the TNF-releasing activity of the two RP-HPLCpools of the precipitate were examined for three batches of thecomposition. Table 31 shows again that for three different batches(B0222, B0221, and B0213), the TNF-releasing activity is principally inthe pool 2 to 21 min. Thus different batches are consistent.

TABLE 31 TNF Releasing Activity of HPLC Precipitate Fractions ofVirulizin Prepared in 80% Acetonitrile 199 Medium Quantity in TNFReleased (pg/ml) Osmolarity Sample Tested Used Well Total −LPS (mOsm)LPS (1) 1X 50 ng 136 ± 62  0 299 LPS (1) 1X 50 ng 81 ± 25 0 306Virulizin: B0222 1X 40 μl 734 ± 276 659 329 Whole 10 μl 119 ± 11  38 303HPLC Precipitate:  2:00-21:25 min 70% 1X 200 μl 20 ± 4  0 354 100 μl 155± 32  74 307 21:25-46:20 1X 200 μl 67 ± 39 0 313 100 μl 78 ± 10 0 310Virulizin: B0221 90% 1X 40 μl 626 ± 90  490 315 Whole 10 μl 62 ± 31 0290 HPLC Precipitate:  2:00-21.45 min 70% 1X 200 μl 20 ± 7  0 365 100 μl170 ± 6  90 329 21:45-46:50 min. 1X 200 μl 45 ± 5  0 336 100 μl 73 ± 150 311 Virulizin: B0213 90% 1X 40 μl 620 ± 123 484 315 Whole 10 μl 80 ±17 0 290 HPLC Precipitate:  2:00-21:30 min 70% 1X 200 μl 6 ± 4 0 393 100[2l 153 ± 45  72 314 21:30-36:40 min 1X 200 μl 182 ± 96  47 312 100 μl78 ± 20 0 310 Note: 1. For 100 & 10 μl samples; 3 patients tested forthese samples. 2. For 200 & 40 μl samples; 4 patients tested for thesesamples. 3. Total TNF released is corrected for TNF release by 199Medium 1X. 4. 199 Medium 1X @ 100 & 10 μl - 306 mOsm, @ 200 & 40 μl -305 mOsm. 5. Osmolarities are averages and not corrected for 199 Medium1X; standard errors are not reported as values are very low. 6. LPSvolume added to wells was 10 μl. 7. Wells contained a total volume of1000 μl. 8. Sample volumes are equivalent. 9. Reconstitution: firstfractions is double distilled water, second in PBS buffer.

In a further experiment, the effect of washing the precipitate with 80%acetonitrile was examined. The point of the experiment was to prove thatthe TNF releasing activity was not being simply trapped. Wholecomposition, 0.04 ml, released 325 pg/ml of TNF. The precipitate poolfrom 2 to 24 min at 0.1 ml released 324 pg/ml of TNF and at 0.05 ml, 3pg/ml. The pool from 24 min to 46 min released no TNF. Likewise thesupernatant pool from 2 to 24 min released TNF at 0.1 and 0.05 ml,whereas the pool from 24 to 46 min had some, but considerably less,TNF-releasing activity on RP-HPLC.

To be certain that the handling of RP-HPLC isolates of the compositionwere not responsible for the presence of TNF-releasing activity, sampleswere prepared in the same fashion but without the composition. RP-HPLCprofiles of PBS and H₂O blanks, their precipitates and supernatants wereessentially free of any peaks.

Using mononuclear cells from identical donors, the samples were testedon leukocytes from the same donors. Whole composition and theprecipitate eluting from 2 to 24 min released TNF, whereas PBS, H₂O ortheir precipitates separated on RP-HPLC had no TNF releasing activity inthe pool from 2 to 23 min. Thus, the precipitate eluting from 2 to 24min causes specific TNF-release.

The precipitate pool of the composition eluting from 24 to 46 minreleased no TNF. The controls of water and PBS showed release of 114 and40 pg/ml, respectively. Thus there was no specific release of TNF fromthe precipitate pool 24 to 46 min.

The supernatant fraction pool 2 to 24 min and 24 to 46 min released 82and 68 pg/ml of TNF, respectively. The water and PBS blank pools fromRP-HPLC released some TNF activity. The water pool 2 to 25 min and 25 to46 min released 149 and 216 pg/ml of TNF respectively, and the PBS poolsreleased 0 and 126 pg/ml respectively.

Thus, both the precipitated and supernatant fraction had TNF-releasingactivity. RP-HPLC separation of the TNF-releasing activity showed thatboth eluted early from RP-HPLC, suggesting that the active componentsare physically very similar if not identical.

Table 32 provides a summary result of further testing, for 80%acetonitrile precipitates and supernatants after RP-HPLC separation,minus the activity in similarly prepared blank samples.

TABLE 32 Releasing activity of Virulizin less release by reconstitutionsolutions. 199 Media Quantity Actual Released (pg/ml) Osmolarity SampleTested Used in Well TNF-a GM-CSF IL-1β (mOsm) Virulizin: B0222 80% 1X 40 μl 178 136 142 310 Whole HPLC Precipitate (min):  2:20-24:10 70% 1X100 μl  75  13  18 314 24:10-46:20 70% 1X 100 μl  0  16  0 292 HPLCSupernatant (min):  2:00-23:55 1X 100 μl  82  86  0 336 23:55-46:20 1X100 μl  0  45  29 316 Note: 1. Data derived from Virulizin SumtableTable 24.4. 2. Virulizin reconstitutition: 1st precipitate fraction inType 1 water, all other fractions in PBS. 3. Whole amounts areunreconstituted and therefore not corrected. 4. Sample volumes areequivalent.

Whole composition releases TNF, GM-CSF and IL-1β in vitro frommononuclear cells at 24 hrs. The 80% acetonitrile precipitate containsthe same releasing activity and most elutes in the early fraction fromRP-HPLC. The supernatant fraction retains releasing activity and mostelutes in the early fraction from RP-HPLC. The supernatant fractionretains releasing activity, but it also elutes in the early elutingRP-HPLC fraction for TNF and GM-CSF. The results suggest that likely thesame component releases the three cytokines. For GM-CSF and IL-1β, thefact that some releasing activity elutes in the late fraction fromRP-HPLC suggest that there may be another substance in the compositionthat can act on monocytes to release GM-CSF and IL-1β.

Physicochemical Analysis SDS Gel Electrophoresis

Having identified that TNF, IL-1β and GM-CSF releasing activity can beprecipitated, in part, by 80% acetonitrile and that much of thereleasing activity elutes early from C₁₈ RP-HPLC, the physicochemicalproperties of the precipitate fraction have been studied and compared tothe whole composition and supernatant fraction of the composition.

FIG. 21 shows an SDS gel electrophoresis of whole composition andprecipitates and supernatants of the composition. In all threeinstances, the composition runs near the SDS front, indicating a lowmolecular weight. The smallest standard used was 14,400 daltons.

Molecular Sieve HPLC

The molecular size of the composition was also examined by determiningits time of elution from a molecular sieve HPLC column. The elutiontimes of whole composition, precipitate and supernatant compared tostandards. All three eluted later than insulin, which eluted at 24.5min. Once again, physicochemical analysis indicates a mol. wt. less than2,400 daltons.

Hydrophilic (Polyhydroxyethyl) HPLC

The TNF-releasing component elutes early. Thus a column with theopposite effect was chosen, a hydrophilic column in the presence oforganic solvents. The ideal eluting conditions for the polyhydroxyethylcolumn is 80% acetonitrile. However, as indicated in the prior Example,some of the substances in the preparation precipitated at thisconcentration. Consequently, the composition was analyzed at a lowconcentration of acetonitrile where the column functions mostly as amolecular sieve column. FIGS. 22 and 23 show the profile of wholesupernatant and precipitate. The front sheet summarizes the elution timefor the different peaks. The elution times indicate the active componetof the composition has a low molecular weight.

Amino Acid Analysis and Sequencing of the Precipitated Component

Two samples were submitted for protein analysis by amino acidcompositional analysis before and after acid hydrolysis: theacetonitrile precipitate and 2-20 min RP-HPLC eluted pool of theprecipitate. The two samples were very similar by comparison of aminoacid content before and post acid hydrolysis. There are, however,significant differences between the amino acid composition (post acidhydrolysis), and the free amino acid content which suggests that peptidebonds were hydrolyzed. The composition of the samples were peculiar inthat they were very rich in glycine plus glutamate/glutamine.

From the foregoing, it may speculated that at least one of the activecomponents is proteineous. Analysis reveals potentially significantquantities of unidentified ninhydrin positive (most likely amino acidcompounds, but other compounds may yield a response) components thatappear to be stable to acid hydrolysis. The principal amino acids per1000 residues in the sample are Asx (asparagine) 143, Thr (Threonine)31, Glx (glutamate) 381, Gly (glycine) 187, and Ala (alanine) 170.

A comparison was made between free and released—(acid hydrolysis) aminoacids. This shows the following amino acids per 1000 residues: Asx(asparagine) 51, Threonine 8.6, Serine 18, Glx (glutamine) 375, Pro(proline) 17, Glycine-(Glyj 429 and Alanine 86.

The ratio of Free/1000 is as follows:

Released/1000 Asx 4.09 Thr 2.03 Ser 1.65 Glx 1.18 Gly 0.37 Ala 1.69

There are 5 unidentified (ninhydrin positive) components. Highlyspeculative assignments are cysteic acid, glucosaminic acid andsarcosine. Also there may be methionine sulfoxide and methioninesulfone. The major unidentified ninhydrin component appears to be freecomponents in the sample.

EXAMPLE 12

Release of IL-1β and IL-8

Table 33 shows that the composition of the invention stimulates humanmononuclear cells in culture to release IL-1β and the 80% acetonitrileprecipitate of the composition releases more than the remainingsupernatant. Whereas the whole composition does not stimulate IL-8release, fractionated composition seems to release some IL-8. Theresults shown in Table 33 are minus the release of IL-1β and IL-8 withmock samples.

TABLE 33 Releasing activity of Virulizin less release by reconstitutionsolutions. 199 Media Quantity Actual Released Osmolarity Sample TestedUsed in Well IL-1β (pg/ml) IL-8 (ng/ml) (mOsm) Virulizin: B0222 80% 1X 40 μl 171  0 304 Whole Precipitate 70% 1X 100 μl 160 71 292 Supernatant90% 1X 100 μl  17 87 335 Note: 1. Number of patients tested: 5. 2.Reconstitution: precipitate in Type 1 water, supernatant in PBS buffer.3. Samples are corrected for release by 1X 199 Medium and LPS: 154, 1065pg/ml, and 68, 294 ng/ml, respectively, for IL-16 and IL-8. 4.Osmolarities for media and LPS are: 311 and 309, respectively. 5. LPSvolume ADDED TO wells: 10 μl. 6. Total volume of wells: 1000 μl. 7.Sample volumes are equivalent.

Physicochemical Characteristics

The composition and its precipitate and supernatant were separated byion-exchange HPLC. Both by AX300 (anion exchange) chromatography and byCMX 300 (cation exchange) chromatography, there was no significantseparation of components. Hydrophobic reverse phase chromatography didnot separate the peaks.

Capillary Electrophoresis

The precipitate was analyzed by capillary electrophoresis. At high pH, aW absorbing peak was observed at 190 nm but completely disappeared at200 nm. There were no significant peaks at 214 nm Uv absorption.

Free amino acid are not visualized unless they are derivatized. It isthought that the W peak at 190 nm is likely a salt.

EXAMPLE 13

The composition was evaluated for stimulatory activity in the following3 indicator systems: 1) Stimulation of lymphocyte DNA synthesis; 2)Induction of lymphocyte-mediated cytotoxic function; and 3) Induction ofmonocyte/macrophage-mediated cytotoxic function. These tests were chosenfor the screen because they measure immunological functions which havebeen shown to be associated with different clinical parameters inpatients with malignant disease. These indicators of immune functionalso can be modulated in cancer patients who are treated with differentbiological response modifying agents such as interferon orinterleukin-2. The results of the initial screening procedure arepresented below.

1) Stimulation of Lymphocyte DNA synthesis: comparison with an optimalstimulating concentration of phytohemaaalutinin (PHA)

Stimulant Counts per Minute Medium   374 PHA 125,817  Composition (#222)1,116 Composition (1:10) 1,021 Composition (1:50)   649

Results: Unlike the prototypic mitogen, PHA, the Composition does notstimulate lymphocytes to undergo blastogenesis and cell division.

2) Stimulation of Lymphocyte-mediated Cytotoxic Function: comparisonwith an optimal stimulating concentration of Interleukin-2 (IL-2)

Stimulant Lytic Units Medium 30.8 IL-2 472.5 Composition (neat) 48.1Composition (1:10) 33.3 Composition (1:50) 44.8

Results: Unlike the prototypic stimulator of lymphocyte cytotoxicfunction, Interleukin-2, the composition does not elicit lymphocytecytotoxicity.

3) Stimulation of Monocyte-Mediated Cytotoxic Function by theComposition: Comparison with Gamma Interferon & Endotoxin (γ-IFN+LPS)

Stimulant (E/T = 20/1) % Cytotoxicity Medium 4.3 IFN + LPS 24.4Composition (neat) 19.7 Composition (1:10) 20.0 Composition (1:50) 11.5

Results: The composition is capable of stimulating peripheral bloodmonocytes to express tumoricidal function in a dose dependent manner.The magnitude of stimulation is comparable to that elicited by theprototypic macrophage activator combination of γIFN+LPS. It is importantto recognize that the action of the composition in these in vitro assaysdid not require the addition of endotoxin as in the case with any othermacrophage activators. If the composition is free of endotoxincontamination, its biological activity in this assay of macrophageactivation would be considered biologically significant.

Monocyte/Macrophage Studies With the Composition

Because the screening procedures demonstrated that the composition doesnot stimulate lymphocyte functions but can stimulate monocyte functions,subsequent studies were aimed at further characterization of themonocyte/macrophage stimulatory activities of this compound. A number ofcomparative studies aimed at determining the dose responsecharacteristics of the composition in stimulating monocyte/macrophagetumoricidal function, were performed as well as testing differentbatches of the compound. The main emphasis of the studies was to testthe capacity of the composition to simulate tumoricidal function inmonocytes and macrophages from different anatomical sites of cancerpatients. The central hypothesis guiding these studies is that thetherapeutic efficacy of any biological stimulator will depend, in largepart, on its ability to elicit tumoricidal function in environmentswhich contain malignant disease. That could come about by directstimulation of resident immune cells in tumor microenvironments.Alternatively, this could come about by stimulation of circulatingimmune cells if those cells were then able to home on sites of malignantdisease and to function in that environment. For these investigations,the following were relied upon: 1) peripheral blood monocytes fromcancer patients and control subjects; 2) alveolar macrophages from lungcancer patients and control patients with non-malignant lung diseases;and 3) Peritoneal macrophages from patients with gynecologicalmalignancies.

1. Dose Response and Different Batch Studies With the Composition

These studies relied on peripheral blood monocytes to test thestimulatory activities of different doses and different batches of thecomposition. Three batches of the composition were provided for testing.These were designated as batch #s 216, 219 and 222. Each batch of thecomposition was tested without dilution (neat), a 1:10 dilution and a1:50 dilution of material. The results are depicted graphically in FIG.24.

Results: Batch #222 and 216 stimulate monocyte tumoricidal function,Batch #219 did not. It appeared that #222 was superior to 216 in thesepreliminary investigations. Batch #222 appears to stimulate equivalentlevels of tumoricidal function at the undiluted (neat) and 1:10 dilutionconcentration with less, but still detectable activity at the 1:50dilution. Batch #216 gave the greatest stimulation of tumoricidalfunction at the undiluted (neat) concentration, with less activity atthe 1:10 dilution and no detectable activity at the 1:50 dilution. Asstated above, Batch #219 did not elicit detectable monocyte tumoricidalfunction at any concentration tested.

2. Tumoricidal Function in Peripheral Blood Monocytes

Tests have been performed on 4 peripheral blood monocyte samples fromcontrol subjects. These tests utilized an optimal stimulatingconcentration of the composition (1:10 dilution of batch #222) and anoptimal stimulating concentration of γ-IFN+LPS. The target cells inthese studies were a cultured, NK-insensitive cell line, the ChangHepatoma.

Stimulant % Cytotoxicity (E/T = 20/1) Medium  5.4 +/− 1 γ-IFN + LPS 18.6+/− 4 Composition 22.3 +/− 6

A test was also performed on 1 monocyte sample from a patient withcervical cancer. This test was important because the patient's own tumorcells were available to be used as target cells in the assay. As before,this test utilized an optimal stimulating concentration of thecomposition (1:10 dilution of batch #222) and an optimal stimulatingconcentration of γ-IFN+LPS. Also, the effector/target cell ratio wasreduced to 15/1 to conserve patient tumor cells.

Stimulant % Cytotoxicity (E/T = 20/1) Medium  5.5 γ-IFN + LPS 14.4Composition 20.9

Results: In the peripheral blood monocytes from control subjects, thecomposition stimulated monocyte tumoricidal function against the ChangHepatoma at a level equal to or greater than the level elicited by anoptimal stimulating concentration of γIFN+LPS. In the peripheral bloodmonocytes from a patient with cervical cancer, the compositionstimulated tumoricidal function against the patient's own tumor cells ata level which exceeded that elicited by γ-IFN+LPS by >30%.

3. Tumoricidal Function in Peritoneal Macrophages From Patients WithGynecological Malignancies

These tests were performed on peritoneal macrophage samples isolatedfrom lavage fluids of 1 patient with cervical cancer and 1 patient withOvarian Cancer. These tests were performed with the patient's own tumorcells as target cells in the assay. As before, an optimal stimulatingconcentration of the composition (1:10 dilution of batch #222) and anoptimal stimulating concentration of γ-IFN+LPS were compared. Also, theeffector/target cell ratio was reduced to 15/1 to conserve patient tumorcells.

Stimulant Cervical Cancer Ovarian Cancer Medium  8.2 0.6 IFN + LPS 29.84.1 Composition 13.2 8.9 (1:10)

Results: These test results highlighted the fact that the local tumorenvironment may be a determinant of the response of immune cells toimmunological activators. In this case of cervical cancer, there was nopathological evidence of malignant disease within the peritoneal cavityand the development of tumoricidal function against the autologous tumorwas better with γIFN+LPS than the composition. In the patient withovarian cancer, there was significant tumor in the peritoneal cavity.The response against the patient's own tumor to γIFN+LPS was minimal atbest, whereas the response to the composition was greater.

4. Tumoricidal Function in Alveolar Macrophages From Lung CancerPatients and Control Subjects

These tests were performed on alveolar macrophage samples isolated frombroncholveolar lavage fluids of a patient with non-small cell lungcancer and 3 patients with non-malignant diseases of the lung. Thesetests utilized an optimal stimulating concentration of the composition(1:10 dilution of batch #222) and an optimal stimulating concentrationof γ-IFN+LPS. The target cells in these studies were the Chang Hepatomacells and the effector/target cell ratio was 20/1.

Stimulant Cancer Patients Control Medium 2.6 +/− 2 19.5 +/− 4 γ-IFN +LPS 10.9 +/− 13  1.2 +/− 5 Composition 5.2 +/− 2 18.6 +/− 8

Results: Alveolar macrophages from lung cancer patients are impaired intheir development of tumoricidal function in response to conventionalmacrophage activators such as γIFN+LPS. These results are consistentwith this observation; they show that the tumoricidal function ofalveolar macrophages from lung cancer patients is greatly reducedcompared to control subjects. They also show that the composition doesnot activate tumoricidal function in either the alveolar macrophages oflung cancer patients or the alveolar macrophages of control subjectswith non-malignant lung diseases.

These preliminary in vitro tests with the composition demonstrate thatit is a macrophage activator. The material provided was able to elicittumoricidal activity in a standard cytotoxicity assay against both anNKinsensitive cell line and against freshly dissociated human tumorcells. The activity elicited was also found to be concentrationdependent in these tests. The capacity of the composition to activemacrophage tumoricidal function in vitro is comparable to that of thebest macrophage activating combination presently available, namely,γIFN+endotoxin. As stated above, the capacity of the composition toelicit this level of tumoricidal function in the absence of endotoxinwould be considered important biologically if the material is free ofendotoxin contamination.

As has been found for other macrophage activators, the activity of thecomposition in stimulating macrophage tumoricidal function varies withthe source of the macrophages. It appears that the composition is anexcellent activator of peripheral blood monocytes being equivalent toγIFN+LPS with normal donors and possibly superior to γIFN+LPS withcancer patient donors. Malignant disease has a significant impact on thedevelopment of monocyte tumoricidal function depending on the activatorused (Braun et al, 1991). One determinant of the biological activity ofdifferent macrophage activators in cancer patients monocytes is thesensitivity of the activator to arachidonic acid metabolism and thesecretion, by the cell of prostaglandins. From these initial studieswith the composition, it appears that activity elicited with thecompound is not sensitive to the inhibitory effects of prostaglandins.If prostaglandin insensitivity can be proven definitively for cancerpatient monocytes stimulated with the composition, this would beconsidered important therapeutically since the effectiveness of manyother biological activators is limited by prostaglandins. Preliminarystudies with 2 specimens indicate that the composition may have goodactivity in peritoneal macrophages, particularly when malignant diseaseis present in the peritoneal cavity.

These preliminary results also illustrate what has been found whencomparing the capacity of different activators to stimulate tumoricidalfunction in peritoneal macrophages of patients with differentgynecological malignancies. In those studies, it was found that thepresence of malignant disease within the peritoneal cavity influencesthe responsiveness of the peritoneal macrophages to specific activators.In patients with cervical cancer, malignant disease is not present inthe peritoneal cavity in general, and thus, the response of the residentmacrophages to γIFN+LPS is normal. When disease is present in thecavity, however, as in the case with ovarian cancer, the response toγIFN+LPS is suppressed. This is related, in part, to changes in thearachidonic acid metabolism of the peritoneal macrophages when malignantdisease is present (Braun et al, 1993). The fact that the compositionapparently can activate tumoricidal function in peritoneal macrophagesfrom ovarian cancer patients against the patient's own tumor cells mayreflect, once again, a mechanism for activation which is independent ofthe arachidonic acid metabolic pathway.

On the other hand, the composition clearly does not activate alveolarmacrophages to become tumoricidal whether malignant disease is presentin the lung or not. Alveolar macrophages from lung cancer patients havebeen found to be inhibited significantly in their development oftumoricidal function when compared to either peripheral blood monocytesfrom the same patients or to control alveolar macrophages from patientswith non-malignant lung diseases (Siziopikou et al., 1991). Thus, thelack of activity of the composition in this case is not surprising.

EXAMPLE 14

The development of tumoricidal function in response to the compositionof the invention and other macrophage activators was investigated inperipheral blood monocytes and peritoneal macrophages from patients withgynecological diseases. More particularly, the patient populationconsisted of 7 patients, 3 with benign disease and 4 with malignantdisease (2 ovarian cancers, 1 endometrial cancer, and 1 cervicalcancer). Samples were removed from patients at the time of surgicalprocedure. Preparations containing peripheral blood monocytes wereisolated from blood samples using the procedure set out in Braun et al.Cancer Immunol. Immunother 32:55-61, 1990 and preparations containingperitoneal macrophages were isolated as set out in Braun et al., CancerResearch 53:3362, 1993. Tumor cell cytotoxicity in response to thecomposition of the invention (1:10 dilution of stock batch 222) andother activators namely gamma interferon (100 U/ml), interleukin-12 (500U/ml), and monocyte-CSF (500 U/ml) was assessed using the monocytecytotoxicity assay described in Braun et al., Cancer Immunol. Immunother32:55-61, 1990.

The results as shown in Table 34, demonstrate that the composition ofthe invention stimulates tumoricidal function in both the peripheralblood monocytes and the peritoneal macrophages from patients withmalignant and non-malignant gynecological diseases. The tumorcytotoxicity elicited by the composition of the invention is equal to orgreater than that elicited by the other biological stimulators whichwere tested.

TABLE 34 The Development of Tumoricidal Function in Response to theComposition of the invention and other Macrophage Activators inPeripheral Blood Monocytes and Peritoneal Macrophages from Patients withGynecological Diseases (3 benign disease, 4 malignant disease) % TumorCytotoxicity (+/− S.E.) at Monocyte/Tumor Cell ratio - 15/1 PeripheralPeritoneal Activator Blood Macrophage Medium  8.6 ± 3 3.1 ± 1 GammaInterferon 18.3 ± 2 9.5 ± 1 Interleukin-12 26.0 ± 4 8.5 ± 2 Monocyte-CSF16.0 ± 2 7.0 ± 2 Composition of the 23.0 ± 6 12.5 ± 2  Invention(Virulizin)

EXAMPLE 15

The effect of indomethacin, a prostaglandin synthesis inhibitor, on thedevelopment of tumoricidal function in response to the composition ofthe invention and other macrophage activators in peripheral bloodmonocytes from cancer patients was also investigated. Samples from thePatients with malignant disease in Example 14 were tested using theassay system as described in Example 14 with the exception thatindomethacin (up to 5 ng/ml) was simultaneously added with thecomposition of the invention, interleukin-12 (500 U/ml), andmonocyte-CSF (500 U/ml).

The results as shown in Table 35 indicate that indomethacin augmentscytotoxicity in response to IFNa, GM-CSF and N-CSF. Thus, thedevelopment of tumoricidal function in response to IFN-y, GM-CSF, andM-CSF was regulated by an indomethacin-sensitive function. In contrast,the development of tumoridical function in response to Phorbol Ester(PNA), IL-12 and the composition of the invention was not regulated byan indomethacinsensitive function i.e. indomethacin did not augmentcytotoxicity in response to the composition of the invention, IL-12 andPNA.

TABLE 35 The effect of Indomethacin, a Prostaglandin SynthesisInhibitor, on the Development of Tumoricidal Function in Response to theComposition of the Invention and other Macrophage Activators inPeripheral Blood Monocytes from Cancer Patients. Activation Conditions #donors % cytotoxicity *IFN-γ 23  11.9 ± 9 *IFN-γ + Indomethacin  25.2 ±17 *GM-CSF 10   7.8 ± 6 *GM-CSF + Indomethacin 17.8 ± 8 *PMA 6  27.3 ±14 *PMA + Indomethacin  22.0 ± 17 IL-12 3 24.7 ± 5 IL-12 + Indomethacin25.6 ± 6 M-CSF 3 14.1 ± 3 M-CSF + Indomethacin 19.0 ± 3 Composition 418.7 ± 6 (Virulizin) Composition 16.4 ± 6 (Virulizin) + Indomethacin

EXAMPLE 16

The effect of prostaglandin E₂ on the development of tumoricidalfunction in response to the composition of the invention in the presenceof indomethacin was investigated. The subject population consisted ofone normal and eight patients (one patient with a pancreatic tumor, twopatients with head and neck tumors, one with endometriosis, and fourwith HIV). Preparations containing peripheral blood monocytes wereisolated from blood samples from the patients using the procedure setout in Braun et al. Cancer Immunol. Immunother 32:55-61, 1990. Tumorcell cytotoxicity in. response to the composition of the invention (1:10dilution of stock batch 222) and indomethacin (up to 5˜g/ml), with orwithout PGE₂(10⁸M), was assessed using the monocyte cytotoxicity assaydescribed in Braun et al., Cancer Immunol. Immunother 32:55-61, 1990.

The results in Table 36 show that 36 pathophysiological levels ofPGE₂(10⁸M) failed to suppress the level of tumoricidal function whichdeveloped in response to the composition of the invention. This is incontrast to the capacity of PGE₂ to suppress tumoricidal function inmonocytes stimulated with γ-interferon (Braun et al, Cancer Research53:3362, 1993).

TABLE 36 The Effect of Prostaglandin E₂ on the Development ofTumoricidal Function in Response to the Composition of the Invention inthe Presence of Indomethacin. % Tumor Cytotoxicity at Monocyte/TumorCell ration = 15/1 Composition Composition (Virulizin) + Composition(Virulizin) + Indomethacin + Diagnosis (Virulizin) Indomethacin PGE₂Normal 19 20 27 Pancreatic 15 14 22 HNSCC  9  8 12 HNSCC 11  3 12Endometriosis 37 37 n.d. HIV  6  7  8 HIV 15 12 19 HIV 21 16 20 HIV 2322 n.d.

EXAMPLE 17

The development of tumoridical function against autologous tumor cellsin monocytes stimulated with the composition of the invention wasinvestigated. Preparations containing peripheral-blood monocytes wereisolated from blood samples from 6 patients (three ovarian cancers, oneendometrial cancer, one cervical cancer and one ENT cancer) using theprocedure set out in Braun et al., 1990. Tumor cell cytotoxicity inresponse to the composition of the invention (1:10 dilution of stockbatch 222) and indomethacin (up to 5˜g/ml), with or without PGE(10⁸ N)was assessed using the monocyte cytotoxicity assay described in Braun etal., 1990, with the exception that the patient's tumor cells were usedin place of the Chang hepatoma cells. The patient's tumor cells weretreated with collagenase and DNase, single cell preparations wereprepared, and the cells were labelled as described in Braun et al. 1990.

The results shown in Table 37 demonstrate that the composition of theinvention is capable of activating the patient's own monocytes to killthe patient's tumor. The composition of the inventon is at least aseffective as the standard biological activators which are currentlybeing used.

TABLE 37 The Development of Tumoricidal Function against AutologousTumor Cells in Monocytes Stimulated with the Composition of theInvention (Virulizin) % Tumor Culture Cytotoxicity Diagnosis Conditions(E/T - 15/1) Ovarian Cancer Medium 2 Composition (Virulizin) 11 OvarianCancer Medium 1 γ-Interferon + LPS 4 Composition (Virulizin) 9 OvarianCancer Medium 0 γ-Interferon + LPS 14 Composition (Virulizin) 11Endometrial Cancer Medium 6 γ-Interferon + LPS 14 Composition(Virulizin) 21 Cervical Cancer Medium 8 γ-Interferon + LPS 30Composition (Virulizin) 13 ENT Cancer Medium 11 γ-Interferon + LPS 12Composition (Virulizin) 25

The experimental results in Examples 14 to 17 indicate that thecomposition of the invention is capable of activating monocytes toexpress tumoricidal function, and it is at least as effective as otheractivators currently being used in the clinical setting; it works in theblood with peritoneal macrophages; and, it appears to not be subject tothe inhibitory effects of prostaglandins, which is one of the principleforms of immunosuppression in patients. The experimental data alsosupports the utility of the composition in the treatment of peritonealand gynecological malignancies.

EXAMPLE 18

Early Toxicity Studies

Toxicity studies were conducted on a variety of animal species. Thestudies are summarized in Table 38. All animals were assessed on thebasis of daily clinical observation while receiving the injections ondays 14, 21 and 30 thereafter. No adverse effects were noted throughoutthe period that injections were administered or during the follow-upperiod (one month for all species except the dogs which were followedfor 4 months).

TABLE 38 Summary of Early Toxicity Studies Animal Quantity Dose WhiteMice 100  0.2 ml i.m. at three day intervals four times Male Wistar 100 2.0 ml i.m. at three day intervals four times Rats Golden 60 1.5 ml i.m.at four day intervals four times Hamsters Guinea Pigs 60 3.0 ml at threeday intervals four times Rabbits 15 5.0 ml i.m. at three day intervalsfour times Cats 10 3.0 ml i.m. at three day intervals six times Dogs 122 ml/kg i.m. given once - observed for four months** Hematologic datacollected every third day for the first 30 days and once monthlythereafter.

A toxicity study was conducted to determine the effect of a single largeintramuscular dose of the composition. Thirteen rats received a singleintramuscular dose of 5 ml/kg of the composition. Three rats wereobserved for 7 days. Ten rats were observed for 14 days followed byeuthanasia and necropsy. No symptoms of toxicity were observed in eithergroup and no gross pathologic findings were observed in the animals thatwere necropsied. Based on these observations the LD₅₀ for intramuscularadministration of the composition in rats was determined to be greaterthan 5 ml/kg. Table 39 summarizes these results.

TABLE 39 Estimation of LD₅₀ in Sprague-Dawley Rats Route of AnimalQuantity Admin. Dose Units/kg* LD50 Sprague 3 male i.m. 5 ml/kg 52.5 >5Dawley Rats 10 i.m. 5 ml/kg 52.5 >5 ml/kg (5 male/ 5 female) *Unitscalculated on the expected range of bioactivity of Lot #B0201 measuredat 10.5 units/ml.

Toxicity Trial In Dogs

In a study conducted by the Ontario Veterinary College, the compositionwas administered to two mixed breed dogs. The protocol is summarized inTable 40. In each case one dose was given in the right leg and thesecond dose 7 days later was given in the left rear leg. Both dogs wereobserved for 14 days after the first injection. Appetite, activity,temperature, pulse rate, respiratory rate were monitored twice dailythroughout the study. Routine urinalyses, hematology and serum chemistryprofiles were performed, pretreatment and 24 hours, 72 hours, 7 days and14 days after the first injection. Neither animal showed signs of painassociated with either injection. There was no evidence of anaphylaxisassociated with the second injection. No abnormalities or changes inphysical or laboratory parameters were observed that could be attributedto the drug. The drug appeared to be well tolerated by healthy dogs.

TABLE 40 Toxicity Study in Dogs Age and Dose 1 Units Dose 2 Units DoseAnimal Weight Calculated* Calculated* Interval Male Adult  5.5 ml i.m.0.6 ml i.m. 7 days Mixed Breed  5 kg 28.6-50.6 Units  3.1-5.5 Units Female 6 months 12.5 ml i.m. 1.3 ml i.m. 7 days Mixed Breed 13 kg65.0-115.0 Units 6.8-12.0 Units

Treatment of Animals With Malignant Neoplasmas

The composition of the invention was used clinically in a veterinaryhospital for the treatment of various malignant tumors in companionanimals. Eleven cats and ten dogs with advanced neoplastic disease thatwas not responding to conventional therapy were treated with thecomposition given intramuscularly in weekly doses. Table 41 summarizesthe individual clinical cases in this study. The number of injectionsranged from 2 to 69, with volumes up to 7.5 ml given into a singleintramuscular site. Protocols of weekly injections allowed forexaminations and careful monitoring of the individual cases withdiagnostic tests determined individually for each case. The cliniciannoted that there was no local irritation nor severe allergic reactions,including anaphylaxis. The clinician and the owners of the animals didnot observe any systemic adverse reactions. The investigators noted someclinical improvements consisting of minor reductions, improved appetiteand activity levels, significant weight gain in a few animals and adecrease in pain and/or discomfort.

TABLE 41 Table 5-5 Summary of Results from Treatment of Animals withMalignant Neoplasms No. Name-Age Species-Sex Diagnosis From-ToInjections Surgeries Results 01 Bandit-13 Canine-M/n Orinasal01.31.87-05.19.87 16 3 Minor Partial Response Fibrosarcoma ProgressiveDisease Euthanasia 02 Bob-5 Feline-M/n Focal Osseous 04.02.87-08.10.8718 11 FIrst recurrence 16 Metaplasia with months all completeOsteosarcomatous response development, Spindle Cell Sarcoma, Feline11.08.88-02.21.90 69 Response with Fibrosarcoma, subsequent treatmentSquamous, evolving to Progressive Recurrent Spindle Cell Disease (tumorbecame Sarcoma - invasive Rapidly invasive) (necropsy diagnosis)Euthanasia 03 J. D.-7 Canine-M/n Oral Amelanotic 03.02.02.03.87 26 3Complete Response. Melanoma, 08.24.87 20 Currently Asymptomatic BenignPapilloma 12.14.87-04.05.88 14 (4 months) Recurrent Round04.04.89-08.01.89 17 Cell Sarcoma 11.09.89-11.30.89 15 241290 4 2502918994 04 Mimi-7 Canine-F/s Invasive 02.27.87-08.10.87 22 5 Stable (NoChange), Fibrosarcoma recurrent Limb amputation. No recurrence 05Goliath-17 Feline-M/n Malignant Melanoma 04.02.87-09.14.87 22 3 InitialMajor Partial Fibrosarcoma Response. Subsequent 11.30.87-07.25.88 31Minor Partial Response. Progressive Disease Euthanasia. 06 Diablo-15Feline-M/n Malignant 05.28.89-06.29.87 5 1 Initial Minor ResponseSquamous Cell Then Progressive Disease Carcinoma Euthanasia 07 Oliver-10Canine-M/n Malignant Round 02.24.89-05.30.89 12 1 Complete Response.Cell Sarcoma Asymptomatic 1 year. 08 Karu-7 Canine-M/n MucinousIntestinal 06.01.87-09.14.87 14 1 Minor Partial Response.Carcinoma-Metastatic Then Progressive Disease. Euthanasia. 09 Puppy-12Feline-M/n Ceruminous 07.22.88-10.04.88 10 1 Minor Partial Response.Gland Adenocarcinoma Then Progressive Disease. Died 10.10.87 10Grandpa-16 Feline-M/n Anaplastic 01.17.89-03.20.89 9 2 Minor PartialResponse. Neoplasma High Grade Then Progressive Disease. MalignancyEuthanasia 03.26.87 11 Sam-7 Feline-F/s Mediastinal 11.02.87-12.21.87 70 Minor Partial Response. Lymphoma Then Progressive Disease. Died12.21.87 12 Pete-3 Feline-M/n Acute Feline 04.13.87-05.11.87 5 0Transient Minor Partial Leukemia Response. Then Progressive Disease.Died 05.05.87 13 Midnight-8 Feline-M/n Feline Leukemia 11.24.87-01.01.882 0 Progressive Disease Euthanasia 01.07.88 14 Stormy-10 Canine-M/nAmelanotic 03.02.87-07.04.87 Complete Response, Melanoma RecurrenceAfter Nine Months, Progressive Disease 15 Penny-10 Canine-F Malignant03.02.87-07.08.87 Partial Response(?) Melanoma Progressive Disease 16Muky-5 Feline-F Anaplastic 02.09.87-06.08.87 6 3 Complete Response,Carcinoma Recurrence After Three Months Stable 17 George-10 Feline-MMalignant 03.02.87-08.04.87 15 1 Minor Partial Response, Melanoma NoChange After 26 Months Stable 18 Simon-13 Canine-M Benign04.02.87-08.31.87 10 1 Minor Partial Response Prostatic Hyperplasia 19Tequila-14 Canine-F/s Malignant 11.24.8-08.09.90 35 1 Minor PartialResponse Intestinal Euthanasia 08.09.90. Adenocarcinoma 20 Sheba-12Canine-F/s Invasive 12.06.89-06.28.90 25 1 Initial Minor PartialOsteosarcoma Response. Then Skull Progressive Disease Euthanasia 21Mesha-14 Feline-F/s Osteosarcoma 02.07.89-05.06.89 13 2 Limb Amputation.Complete response. No recurrence or metastases 1 yr.

The clinical results are summarized as follows: Six animals (3/10canines and 3/11 felines) experienced complete response. One animal(1/11 felines) had initial major partial response. Eleven animals (5/10canines and 6/11 feline) experienced minor partial response. One animal(1/10 canines) remained stable and one animal (1/11 felines) did notrespond. Table 42 provides definitions of each treatment. The clinicalexperience in animals suggested a potential role for the composition inthe treatment of malignant neoplasms.

TABLE 42 Definition of Treatment Responses Response Definition CompleteResponse Disappearance of all clinical evidence of active tumors. Thepatient must be free of all known disease as determined by twoobservations not less than four weeks apart. Partial Response MajorWhere there is a greater than 50% reduction in the sum of the product ofthe perpendicular dimension of all measurable tumor with no new lesionsappearing elsewhere. Minor Where there is a 25-50% shrinkage in the sumof the products of the perpendicular diameters of all measurable tumors;or subjective responses such as improvement in performance status,appetite and feeling of well being; or tumor necrosis or lysis as seenon ultrasound, x-rays, or changes in consistency and character of thetumors suggesting a decrease in adhesions and an increase in tumormobility. Stable Disease Less than 25% increase or decrease in the sizeof one or more measurable lesions without tumoral lysis, or appearanceof new lesions. Progressive Disease Increase of 25% in the size of oneor more measurable lesions without tumoral lysis, or appearance of newlesions.

EXAMPLE 19

Preliminary Clinical Trials

Patients with untreatable tumors were treated with 0.11 ml per kilogramof the composition of the invention as prepared in accordance with themethods set out in Example 1. The composition was given intramuscularlyevery three to five days. Of the 58 patients treated there wasabsolutely no significant toxicity. In the 37 evaluable patients threepatients had minor responses i.e. tumor shrinkage of between 25 and 50percent, and five patients had stable disease of at least eight weeks induration. The most interesting results were in the pancreatic patientswho seemed to have the most encouraging results. Of the seven patientsone patient had the disease stabilization for a full 11 months. And asecond patient with extremely advanced disease had disease stabilizationfor four months. It was based on these results that carcinoma of thepancreas was selected for a basic study of the composition of theinvention. The objective of the study was to determine the safety andefficacy of the composition in this group of patients.

The treatment consisted of the composition of the invention 0.11 ml perkilogram with a minimum dose of 7.5 ml given as a single deepintramuscular injection to the gluteus maximus. Patients received thetreatment three times weekly during the first week and then twice a weekuntil disease progression. In all, 22 patients were enrolled in thisstudy and all were evaluable for toxicity. Only 17 patients wereevaluable for efficacy. With a total of 570 injections there was notoxicity of any kind reported in either local or systemic. There werealso no objective responses. Six patients however had stable disease forthree months or longer but the rest of the patients progressed withinthe first three months. The median survival was eight months from thetime of diagnosis. There was a median survival of four months from thefirst injection. There were three patients who had stable disease forlonger than six months. One patient, a 75 year old man relapsed withliver metastasis 18 months after a liberal procedure. He remainedabsolutely stable on the composition for eight months beforeprogressing. A 71 year old woman with unresectable disease remainedstable for at least ten months and continued to work full time. A 64year old woman who relapsed regionally four months after the procedurewas stable for at least eight months.

A fourth patient who had inoperable carcinoma of the pancreas and couldnot be enrolled in the study because tissue could not be obtained for apathologic diagnosis, was stable for almost a year although his tumorprogressed despite higher doses of the composition. In summary, thecomposition has no site of toxic activity against pancreatic cancer atthis dosage schedule. There was a suggestion of temporaryanti-proliferative activity in a minority of cases with this diseaseusing the composition.

EXAMPLE 20

Pancreatic Cancer Clinical Studies

A Phase II trial with the composition of the invention was begun forpatients with measurable, biopsy-proven pancreatic cancer. Thecomposition was administered as a 7.5 ml (0.11 ml/kg) intramuscularinjection 3 times weekly for 1 week then twice weekly until diseaseprogression. Details of the study are set out below.

Method

Treatment consisted of the composition prepared as in Example 1, 0.11ml/kg (minimum dose 7.5 ml) administered with a single deepintramuscular injection to the gluteus maximus, alternating buttockswith each dose. Patients received 3 injections during the first weekfollowed by twice-weekly injections until tumor progression.

Response was defined using standard criteria. Miller et al., Cancer1981; 47:207-214). A complete response (CR) was defined as completedisappearance of all evidence of disease for at least 4 weeks. A partialresponse (PR) was defined as a ≧50% reduction in the product of the twolargest perpendicular diameters of the largest measurable lesion, withno new lesions or progression of any lesion, for at least 4 weeks.Progressive disease was defined as a 25%, or more increase in the sizeof one or more measurable lesions or the appearance of new lesions.Disease not meeting criteria for response or progressive disease wastermed stable disease.

Results

A total of 22 patients were enrolled in the study, but five patientswere considered inevaluable for efficacy. There were no complete orpartial responses. Three patients had disease progression within thefirst month. Six patients had disease stabilization for more than 3months (3.5, 3.5, 5, 8, 12+, 14+). Median survival for the entire groupwas 8 months from the date of diagnosis and 5 months from the start oftreatment. One patient with biopsy-proven liver metastases and a CEA of37 ng/ml (normal <3 ng/ml), had absolute stabilization of the livermetastases and CEA for 8 months. One had stable disease for 5 months.One patient had disease relapse in her pancreatic bed 4 months after aWhipple procedure and was been stable on the composition for at leastone year, with the exception of a slowing rising CEA. A third patienthad a percutaneous stent inserted and continued to work full-time for atleast 14 months with no evidence of tumor progression.

All 22 patients were evaluable for toxicity, having received a total ofover 500 injections. None developed any clinical or laboratory evidenceof drug-related toxicity. There was no detrimental effect on Quality ofLife which generally parallelled disease activity. No significantchanges in total white blood cell counts, absolute lymphocyte counts onserum immunoglobulins were seen.

Survival curves representing the survival times from diagnosis and fromtreatment initiation are presented in FIGS. 13 and 14, respectively. Forcomparison, an historical survival curve for Gudjonsson (1987) has beensuperimposed in FIG. 13. Another example of a comparable historicalsurvival curve may be found in Bakkevold, Petterson, Arnesjo andEspenhaug (1990).

The results of the survival analyses are summarized in Table 43. Themean survival time for diagnosis was 281 days (FIG. 13). The mediansurvival was 182 days (approximately 5 months). For comparison,Gudjonsson (1987) reported the mean survival of his 188 surgicalpatients as 208 days with a media survival of 120 days. The meansurvival time from treatment start was 166 days (FIG. 14). The mediansurvival was 133 days (approximately 4 months and 1 week).

TABLE 43 Protocol CO2-104 Survival Estimates Patient Mean SurvivalStandard Median Survival Population (days) Deviation Survival (days)From Protocol 281 203 182 diagnosis CO2-104 patients Protocol 304 157219 CO2-104 evaluable patients From Protocol 166 135 133 treatmentCO2-104 start patients Protocol 220 132 146 CO2-104 evaluable patients

Survival times were also estimated among a subset of evaluable patientswho had each received at least 13 injections. Fourteen of the 22 patientwere evaluable. Among these patients (Table 43), the median survivalfrom diagnosis was 219 days (approximately 7 months and 1 week). Themedian survival from treatment start was 146 days (approximately 5months).

EXAMPLE 21

Clinical Trials re Malignant Nelanoma

Advanced malignant melanoma was defined to include all stage III or IVpatients and all loco-regional or distant relapses occurring afterprimary treatment. The standard treatment by which all other treatmentsare judged is DTIC (dacarbazine) which has a reported response rate ofabout 15%. The median response is 3-6 months, and carries with it severenausea and vomiting, and a potentially lethal side effect of acute livernecrosis by thrombosis of the hepatic veins. This treatment fails toshow any definitive survival advantages.

This study was conducted to determine the safety and efficacy of thecomposition of the invention and to determine its effect on survival andon quality of life, when used in patients with advanced malignantmelanoma. The study, was a non-comparative, multicenter trial.

An initial dosing schedule of 7.5 ml injections of the composition ofthe invention intramuscularly 3 times. per week was used. After no organor marrow toxicity was observed, the loading schedule was increased todaily injections for 15 days, followed by maintenance of 3 injectionsper week. Subsequently the loading dose was increased to 30 days.Duration of treatment was 36 weeks and then reduced to 16 weeks, afterwhich patients were given the option of entering a continuationprotocol.

Thirty-three patients with advanced melanoma were included in the studypopulation (17 females and 16 males), ranging in age from 17 to 85 yearsof age. 64% had been previously treated and 36% were untreated. Of the33 patients included in the study population, twenty five wereevaluable. The Karnofsky Performance Status (baseline) was in the rangeof 40-100%, median 80%. Eleven patients were alive at the end of thestudy period and five of these were under treatment.

A minor partial response was observed in 16/33 patients (48%). Onepatient had a reduction of 33% in the lungs, six patients had painreductions and eight patients gained more than 1000 grams in weight formore than a month (Range 1000-2600 grams). A stable condition wasobserved in 19/33 patients (58%) (Range 60-170 days, median 77 days).

FIGS. 15, 16 and 17 show the survival of patients treated with thecomposition of the invention compared to historical controls, measuredas survival from diagnosis of metastases/recurrence in days. The solidline represents the survival curve for patients treated with thecomposition of the invention and the broken line represents thehistorical survival curve (Balch, C. M. et al., Cutaneous Nelanoma, 2nd.ed. 1992, Chps. 14 and 39, pp. 165-187 & 499-508, Lippincott Co.,Philadelphia, Pa.). The survival of all patients treated with thecomposition of the invention, including patients with one to over threetumor sites, is shown in FIG. 15. Survival of patients with two tumorsites and with three or more tumor sites is shown in FIGS. 16 and 17respectively.

The group of all patients treated with the composition of the inventionhad a 39% survival (Kaplan-Meier estimation) at one year. The survivalrate at one year for all advanced malignant melanoma (AMM) patients isapproximately 11% in historical controls (matched by number of tumorsites). The group had a median survival of 315 days compared to thehistorical median of 89 days.

With two tumor sites the one year survival was 49% in the patientstreated with the composition of the invention, as compared with 13% inhistorical controls. This group had a median survival of 360 dayscompared to the historical median of 120 days. With three or more tumorsites the one year survival was 31% in the patients treated with thecomposition of the invention, as compared with 0% in historicalcontrols. The group with three or more tumors had a median survival of205 days compared to the historical median of 60 days.

Quality of life was assessed by weight gain, performance status(Karnofsky), Quality of Life Index (Spitzer) and pain scale (LinearAnalogue). weight gain over time is shown in Table 39.

TABLE 44 Number of 1st 2nd 3rd 4th 5th 6th patients/ month month monthmonth month month evaluable 11/25 12/25 4/25 4/25 1/25 1/25 Percent 44%48% 16% 16% 4% 4% (%) Range 100- 200- 100- 100- (gr) 2400 6000 1000 2000Average 900 1480 525 775 100 2000 (gr)

The Karnofsky and Spitzer scales are both subjective and were found toapproximately agree in each individual. Fifteen patients reported nochange in these parameters. Four patients showed fluctuations whichlater returned to previous levels. One patient had a decrease (from40-20%).

The results of pain evaluation showed that in six patients by week 4 thepain dropped from 5 (worst possible) to 2 (moderate) or 0 (no pain). Onepatient had a drop in pain from 3 to 0. One patient with hepaticmetastasis had pain reduction to 0 and stabilisation for 11 months. Ninepatients who entered the study with 0 pain maintained that levelthroughout the study. Five patients had a moderate (2 unit) increase inpain. Three patients had transient pain increases (1 to 2 units) duringthe second or third month.

Out of 1734 injections administered to 33 patients, 21 patients had noadverse drug reactions. Fourteen adverse drug reactions were reported in12 patients. The adverse drug reactions usually occurred at weeks 4 or 8and were mild to transient, and most frequently were a low grade fever.

The difference in survival between the historical groups and theprotocol groups treated with the composition of the invention suggests asurvival benefit for patients treated with the composition of theinvention. The cancer seemed to stabilize in 19 patients. All patientstreated for ANK were included in the survival data. Also included were21 previously treated patients (many clinical trials require untreatedpatients, because of the poor prognosis of failed previous treatments).The tumor burden was high (82% had more than one metastatic site).

The survival and quality of life data suggest that most patientsreceived some benefit from the treatment. Eleven patients were stillalive at the end of the study period and of those 11, 5 continuedtreatment.

EXAMPLE 22

Pathology Protocol Malignant Melanona

The following is a report of a 73 year old female with progressivemalignant melanoma of the hard palate and gums. FIG. 25 shows two viewsof malignant melanoma as seen under the microscope. In FIG. 25a, lookingfrom top to bottom, one can see the epithelial layer with accompanyingkeratin, beneath which the malignant cells start to became moreapparent. These melanoma cells can be seen to be rounded or oval, withan abundant eosinophilic cytoplasm, and pleomorphic hyperchromaticnuclei. These cells have substituted the normal submucosal tissue. Theblood vessels which are seen appear normal, and there is a paucity ofany kind of inflammatory/immune response as would be represented by thepresence of leukocytes (polymorphonuclear and mononuclear cells). Thisis an example of tumor tissue which is thriving, i.e. the tumoralarchitecture is intact.

In FIG. 25(b) a tumor tissue sample is shown from the same patient, whohad been treated with the composition for two months. Starting from topto bottom, one can see that the continuity of the epithelium has beendisrupted by a necrotic process. This necrosis, while common in thecenter of any tumor that has reached a critical mass, is rarely seen onthe periphery, especially in malignant melanoma, and is a sign that thehost's immune response is mounting an attack against the tumor.Throughout the photo are a massive number of cells different from theoriginal tumor cells. These are the immune cells-neutrophils,lymphocytes, macrophages—which have orchestrated the disruption of thetypical tumoral architecture. The blood vessel walls have become denselyinfiltrated with a large number of host immune cells (arrow). Thiscellular infiltrate subsequently will cause the destruction of the bloodvessel, which in turn prevents the tumor from receiving its supply ofnutrients and oxygen (ischemic necrosis). This immune response whichcontributed to the tumoral disruption seen in this patient's tissueslide is consistent with reported changes known to be brought about byTNF (tumor necrosis factor) and with the results of the work describedin the previous examples.

The immune response demonstrated in the after treatment with thecomposition slide (FIG. 25b) strongly links the in vitro TNF immunemodulation by the composition with known in vivo anti-tumoral TNFeffects.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

EXAMPLE 23

Isolation of Active Fractions

A 300 ml sample of the composition was evaporated to dryness on arotovap in which the temperature of the bath did not exceed 40° C. Inorder to ensure that the solution remained basic during the evaporation,5 drops of a concentrated ammonium hydroxide solution was added everyhalf hour to the composition until the evaporation was complete. Theresulting residue had a weight of 11.6 g.

20 ml of a 10% concentrated ammonium hydroxide in methanol solution wasthen added to 2 g of the above residue. The insoluble material wasfiltered off and the filtrate was chromatographed through 101.93 g of 60Å flash silica gel in a column with dimensions of 5 cm×12.5 cm. Thesolvent system used was 10% concentrated ammonium hydroxide in methanolsolution. The column was run at a pressure of 10 p.s.i. and a flow rateof 11 ml/min. After 100 ml of solvent had passed through the column,twelve 20 ml. fractions were collected. The collection of thesefractions correlated to the appearance of an off-white band that wasquickly moving down the column.

Thin layer chromatography (TLC) of these fractions was run on silica gelplates in a 10% concentrated ammonium hydroxide solution in methanol andvisualized with a ninhydrin spray. Fractions having similar TLC profileswere combined, resulting in the following fraction combinations, whichwere dried on a rotovap.

Volume Through Column to Obtain Fractions Fraction Yield (g) 1-4 100-1800 5-6 180-220 0.1175 7-8 220-260 0.1969  9-10 260-300 0.0151 11-12300-340 0.0053 Fractions 5-6, 7-8 and 9-10 had a positive reaction withninhydrin at an R_(f) value of 0.81.

EXAMPLE 24

Fractions 5-6 and 9-10 from Example 23 were tested in vitro foranti-proliferative effect (in accordance with the procedure of Example4) and TNF stimulation (in accordance with Example 9). The results areshown below:

FRACTION ASSAY ACTIVITY 5-6 Anti-Proliferative Effect 11.57 units/mg 5-6TNF Stimulation - LPS 50 pg/mg  9-10 Anti-Proliferative Effect 2.6units/mg  9-10 TNF Stimulation - LPS 1814 pg/mg

Thus, fraction 5-6 was an active anti-proliferative, and fraction 9-10was an extremely active TNF stimulator, and a moderately activeanti-proliferative.

EXAMPLE 25

Samples of Fraction 5-6 was analyzed by Electron Impact MassSpectroscopy (EI MS) and Electrospray Mass Spectroscopy to identifyspecific compounds likely to be present in the fraction. TheElectrospray MS was performed on a Perkin-Elmer Sciex API-IIIspectrometer, using 5% acetic acid in water as the solute. In someinstances, methanol was added to aid dissolution. The EI MS using adirect insertion probe was performed on a VG Analytical model ZAB-SEspectrometer using glycerol as a matrix, and using a DCI probe on aKratos Analytical Profile Mass Spectrometer.

A review of the resultant spectra indicated that the following compoundswere likely present in Fraction 5-6: phosphocholine, taurocholic acid,choline-stearic acid diglyceride, stearic acid, stearic aciddiglyceride, palmitic acid-stearic acid diglyceride, and asphingosine-oleic acid conjugate.

EXAMPLE 26

100 ml of the composition was acidified with 4 ml of a 1N HCl solutionsuch that the pH of the composition was equal to 3. The composition wasthen extracted with three 100 ml. portions of HPLC gradedichloromethane. The dichloromethane fractions were then combined anddried over a small amount of anhydrous sodium sulfate. Thedichloromethane solution was then filtered through paper into a roundbottom flask and evaporated to dryness on a rotovap to yield 0.0049 g ofa brown film.

0.0017 g of this film was dissolved in a 214 ppm NH₃.H₂O solution at pH7, and was screened for anti-proliferation activity as is set forth inExample 4. This screen revealed that the solution was an activeanti-proliferative, with an activity of 14 Units/mg.

EXAMPLE 27

Example 23 was repeated on a larger scale, as follows. 10 ml of aconcentrated ammonium hydroxide solution was added to 900 ml of thecomposition and the resulting solution evaporated to dryness on arotovap in which the temperature of the bath did not exceed 40° C. Inorder to ensure that the solution remained basic during the evaporation,5 drops of a concentrated ammonium hydroxide solution was added everyhalf hour to the composition until the evaporation was complete, leavinga residue.

150 ml of a 10% concentrated ammonium hydroxide in methanol solution wasthen added to the total residue. The solution was sonicated for 15 min.and the insoluble material was filtered off. The filtrate waschromatographed through 1695 g of 60 Å flash silica gel in a column withdimensions of 30 cm×12 cm. The solvent system used was 10% concentratedammonium hydroxide in methanol solution. The column was run at apressure of 6 p.s.i. and a flow rate of 30 ml./min. The results of thecolumn are summarized in the table below.

Volume of each Fraction # fraction (ml.) Observations  1 550 colorless 2 450 colorless  3 400 colorless  4 150 colorless  5 100 colorless 6-7 75 colorless  8-13  50 colorless 14  50 tan colored solution begins toelute 15-35  50 tan colored solution 36-40  50 colorless

TLC was run on silica gel plates in a 10% concentration ammoniumhydroxide solution and visualized with a ninhydrin spray. Fractionshaving similar TLC profiles were combined, resulting in the followingfraction combinations, which were dried on a rotovap.

Volume Through Column to Obtain Fraction # Fraction Yield (g) Comments 3 1000-1400 0.0504 white powdery solid 4-5 1400-1650 0.0855 whitepowdery solid 6-8 1650-1850 0.1555 white powdery solid  9-12 1850-20500.3014 white powdery solid 13-14 2050-2150 0.3595 white powdery solid15-16 2150-2250 0.6914 slight brown color - solid is tacky 17-182250-2350 1.0284 tan color - solid is clumpy 19 2350-2400 0.3432 tancolor - solid is clumpy 20-23 2400-2600 1.1531 brown color - solid isclumpy 24-30 2600-2950 0.8517 brown color - solid is clumpy 31-342950-3150 0.0813 brown oil

All fraction combinations from 15-16 through Fraction 31-34 had apositive reaction with ninhydrin at an R_(f) value of 0.87, a value verysimilar to the R_(f) value for the active fractions of Example 23.Fractions 24-30 and 31-34 had an additional positive reaction withninhydrin at an R_(f) value of 0.85.

EXAMPLE 28

Fractions 4-5, 15-16 and 17-18 were tested in vitro foranti-proliferative effect (in accordance with Example 4) and TNFstimulation (in accordance with Example 9). The results are shown below:

Fraction Assay Activity 4-5 Anti-proliferative 4.7 units/mg Effect 4-5TNF Stimulation — 15-16 Anti-proliferative 4.5 units/mg Effect 15-16 TNFStimulation — 17-18 Anti-proliferative 3.9 units/mg Effect 17-18 TNFStimulation —

Thus, fractions 4-5, 15-16, and 17-18 showed anti-proliferativeactivity, but no TNF stimulation activity. Elemental analysis of theabove fractions showed them to be high in NH₄Cl , which inhibits TNFproduction.

EXAMPLE 29

Sample of fractions 15-16 and 24-30 was dialyzed and then was analyzedby mass spectroscopy, using the methods described in Example 25.Undialyzed samples from fractions 17-18 and 24-30 were also analyzed. Areview of the resultant spectra indicated that the following compoundswere likely present: glycocholic acid, a trihexosamine trimer, andtaurocholic acid (Fraction 15-16); stearic acid, and a hexosamine dimer;and glycocholic acid (Fraction 24-30).

EXAMPLE 30

The composition was dialyzed in separate dialysis tubing as follows:

100 ml of the composition was placed inside a Spectra/Por™ CE membranetubing which had a molecular weight cut off of 100. The ends of thetubing were sealed with clips and the tubing was placed into a stirredbath of 10 L of distilled water. The dialysis was monitored daily byremoving 1 ml. of solution from the dialysis tubing and adding 3-4 dropsof a 1/10 N silver nitrate solution. The presence of chloride indicatedthat the dialysis was not complete. If the dialysis was not complete thebath was replaced with fresh distilled water. Dialysis completionoccurred after 3-4 days. After dialysis was complete, the dialyzedmaterial was dried on a rotovap to yield an average of 0.3 mg of solidper ml of original volume.

A sample of the solid material was then dissolved in HPLC grade water,and TLC was run on silica gel plates in a 10% concentrated ammoniumhydroxide solution in methanol, and visualized with a ninhydrin spray. Apositive reaction with ninhydrin was obtained at an R_(f) value of 0.83.

EXAMPLE 31

A sample of the solid material from Example 30 was also analyzed by massspectroscopy, using the methods described in Example 25. A review of theresultant spectra indicated that the following compounds were likelypresent: a sphingosine-oleic acid conjugate, diacetyl sialic acid, afucose-hexosamine dimer, deoxyglycocholic acid, taurocholic acid, asialic acid-fucose dimer, and a di(fucose)hexosamine trimer.

EXAMPLE 32

Previous results indicated that the active components of the composition(at least according to a TNFα release assay) were present in the unboundfractions (void volume) after reversed-phase high performance liquidchromatography (RP-HPLC) on a C18 μBondapack column. As well, most ofthe mass (70%) of the composition extract, which was loaded onto a C18μBondapack column, eluted in the void volume. These results suggestedthe active components of the composition are, very likely, very polar oreven ionic molecules.

To further examine the above results, purification of the activecomponents by ion-exchange chromatography was performed. Negativelycharged active components (assessed by its anti-proliferation affect ontumor cells and not on normal cells, and as wall by its TNFαrelease-inducing activity), if present, would thus become bound to ananion exchange resin.

Experimental Procedure

10 ml of total virulizin extract was loaded onto an anion-exchangechromatography column (Bio-Rad AG-1, hydroxide form, total resin wetvolume was 10 ml (column dimensions 1.5 cm×6.0 cm), equilibrated withMillipore deionized water). The volume of resin used was calculated tobe sufficient for the binding of all the anions present in the extract.The unbound fraction was collected and reloaded onto the column in orderto maximize the binding to the resin. The unbound fraction from thissecond passage was collected and saved. Any unbound material remainingon the column's void volume was removed by washing with deionized water(2×20 ml). Bound molecules were eluted with a step gradient of ammoniumbicarbonate (NH₄HCO₃) (20 ml/step).

The elution steps were   0 M 0.1 M 0.2 M 0.3 M 0.4 M 0.5 M 0.6 M 1.0 M1.5 M

EXAMPLE 33

Samples from all the fractions of Example 32 were analyzed foranti-proliferation activity and TNF stimulation activity, in accordancewith the procedures of Examples 4 and 9, respectively.

The results are shown below:

Anti-proliferative TNFα release-inducing Sample activity (U/mg)activity-LPS (pg/ml)   0 M 0 35 0.1 M 0 −79 0.2 M 0 −76 0.3 M 0 0.4 M2.5 0.5 M 555.6 0.6 M 0 107 1.0 M 0 105 1.5 M 0 189

The results from the activity assays show that TNF productionstimulation was found in the 0.6 M, 1.0 M, 1.5 M fractions.Anti-proliferative activity was found in fractions 0.4 M (minoractivity) and 0.5 M (major active fraction).

EXAMPLE 34

Thin layer chromatography analysis of the active fractions revealed amixture of several components. A sample of the 1.0 M fraction fromExample 32 was analyzed by mass spectroscopy in accordance with Example25. A review of the spectra generated suggested that the followingcompounds may be present: a sialic acid-glycerol dimer, cholesterolsulfate, and taurocholic acid.

EXAMPLE 35

Reversed Phase (C18) HPLC analysis was performed on a sample of thecomposition in accordance with the procedure of Example 2, except that(1) a Phenomenex WP60009-C18 column, 250×4.6 mm, was used, (2) thesample was lyophilized and then reconstituted in 0.1% trifluoroaceticacid (TFA) in water, and (3) 150 μl of the reconstituted sample wasapplied to the column.

Various fractions of eluent were collected, including a fraction whicheluted at approximately 2.40-3.40 minutes after the reconstituted samplewas applied to the column.

EXAMPLE 36

A sample of the fraction eluting at approximately 2.40-3.40 minutes fromExample 35 was analyzed three times for TNF stimulation activity inaccordance with the procedure of Example 9. The following results wereobtained.

Assay # TNF Stimulation - LPS 1 259 pg/ml ± 107 pg/ml 2 311 pg/ml ± 14pg/ml  3 572 pg/ml ± 176 pg/ml

EXAMPLE 37

A sample of the fraction eluting at approximately 2.40-3.40 minutes fromExample 35 was subjected to tandem column reversed-phase (C18) HPLC asfollows. The column from Example 35 was used in tandem with a Phenomenexprime-sphere HC-C18 column, 250×4.6 mm. The sample was lyophilized,reconstituted in 0.1% TFA in water (Buffer A) and 150 μl ofreconstituted sample was applied to the column. Buffer A was run fortwenty minutes, then a linear gradient of 0-80% of 0.1% TFA inacetonitrile (Buffer B) was run for 35 minutes. At the end of thisperiod, 80-0% Buffer B was run for 5 minutes. Flow rate was 0.9 ml/min.Six eluent fractions were collected, at the following approximate timesfrom injection:

Fraction # Time (min.) 1  5.6-6.25 2 6.25-6.6  3 6.6-7.1 4 7.1-8.2 58.8-9.6 6 14.7-16  

EXAMPLE 38

A sample of Fraction 1 (“1”) and a sample of Fraction 2 (“2”) fromExample 37 were lyophilized and reconstituted in 214 ppm NH₃.H₂O. Thesereconstituted samples were then analyzed for anti-proliferative effectin accordance with the procedure of Example 4. The following resultswere obtained:

Sample Anti-proliferative Effect 1 17.8 unit/ml 2  0

EXAMPLE 39

Samples from each of the six fractions of Example 37 were analyzed bymass spectroscopy in accordance with Example 25. A review of theresultant spectra for the six fractions indicated that the followingcompounds were likely present: taurocholic acid, a sialic acid-glyceroldimer, NaCl, trimethylamine, methylethylamine, and propylamine.

EXAMPLE 40

Anti-proliferative effect, according to the method of Example 4, wasmeasured for the following three samples: (1) 10-11 mg taurocholic acidin 2 ml of H₂O; (2) 214 ppm NH₃.H₂O; and (3) 0.7 mg of taurocholic acidin 4.0 ml of 214 ppm NH₃.H₂O. Neither sample (1) nor sample (2) had anydetectable anti-proliferative effect. Sample (3), however, had ananti-proliferative effect of 14 units/mg.

This result indicates that bile acids, such as taurocholic acid, incombination with ammonium ions, exhibits anti-proliferative activity atconcentrations below that which the components, tested individually,show no activity. Thus, the combination of these two components,apparently synergistically, affects anti-proliferative activity.

What is claimed is:
 1. A process for preparing a composition for use asan immunomodulator comprising small molecular weight components of lessthan 3000 daltons, and having the following properties: a) is extractedfrom bile of animals; b) is capable of stimulating monocytes andmacrophages in vitro; c) is capable of modulating tumor necrosis factorproduction; d) contains no measurable level of IL-1, IL-1b, TNF, IL-6,IL-8, IL-4, GM-CSF or IFN-gamma; e) shows no cytotoxicity to humanperipheral blood mononuclear cells; and f) is not an endotoxin, saidprocess comprising (a) mixing bile from an animal with an equal volumeof an alcohol to produce a bile/alcohol solution; (b) separating out thealcohol soluble fraction and isolating a solution substantially free ofalcohol; (c) removing bile pigments from the solution to obtain acolorless liquid; (d) treating the colorless liquid to substantiallyremove any residual alcohol; (e) extracting the colorless liquid withether and isolating the aqueous phase; and (f) removing residual etherfrom the aqueous phase.
 2. A process as claimed in claim 1 wherein priorto step (e) the colorless liquid is concentrated to about one eighth ofthe volume of the bile/alcohol solution and after step (f) the aqueousphase is concentrated so that it is one tenth of the volume of thebile/alcohol solution.
 3. A process for preparing an immunomodulatorcomposition comprising (a) mixing bile from an animal with awater-soluble solvent to produce a bile/solvent solution; (b) isolatingan aqueous solution substantially free of solvent from the bile/solventsolution; and (c) removing bile pigments from the substantiallysolvent-free solution to obtain a colorless liquid.
 4. The process ofclaim 3 wherein the water soluble solvent is an alcohol.
 5. The processof claim 4 wherein the bile from an animal is mixed with an equal volumeof an alcohol.
 6. The process of claim 3, further comprisingconcentrating the colorless liquid to about one-eighth the originalvolume of the bile/solvent solution.
 7. The process of claim 3, furthercomprising concentrating the colorless liquid to about one-tenth theoriginal volume of the bile/solvent solution.